Electrochemical detection of bacterial and/or fungal infections

ABSTRACT

The present disclosure relates to methods and devices for amplifying a plurality of targets in a single PCR run while distinguishing between clinically relevant amplification and amplification from other sources such as from background contamination. The methods and devices further enable discrimination between gram-positive, gram-negative and fungal infections as wells as identify antimicrobial resistance genes. When applying the methods and devices of the invention, the species or genus of an infection(s), and genus of a fungal co-infection(s) or category of bacterial (gram-positive or negative) co-infection(s) are identified. Species identification of co-infections can also be achieved. Further, when applying the methods and devices of the invention, organisms which are likely to be contaminating organisms from a blood draw are identified.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 15/828,074,filed on Nov. 30, 2017, which is a continuation of U.S. patentapplication Ser. No. 15/686,001, filed on Aug. 24, 2017. The priorapplications are incorporated herein by reference their entirety.

The invention relates to the field of molecular diagnostic methods, inparticular, microfluidic devices for the detection of target analytes.

INCORPORATION BY REFERENCE

This application is related to U.S. Pat. Nos. 7,820,391, 7,560,237,6,013,459, 6,740,518, 6,063,573, 6,600,026, 6,264,825, 6,541,617,6,942,771, 6,432,723, 6,833,267, 7,090,804, 7,935,481, 7,172,897,6,753,143, 6,518,024, 6,642,046, 6,361,958, 6,602,400, 6,824,669,6,596,483, 6,875,619, 7,863,035, 9,598,722 and U.S. patent applicationSer. Nos. 12/914,257, 14/206,871, 14/206,932, 15/026,314, 14/538,533,14/206,817, 14/538,602, 14/206,867, 14/206,903, 14/062,860, and14/538,506, the respective disclosures of which are hereby incorporatedby reference.

BACKGROUND OF THE INVENTION

In North America, the most common causes of a sepsis are bacteria suchas Escherichia coli or Staphylococcus aureus. In addition to bacterialinfection, fungal infections have in recent times become a significantcause of the disease. Fungal infections tend to be associated withhigher rates of death. Only approximately 5% of fungal caused cases ofsepsis are identified during the disease due to the poor diagnosticmethods available. Recent studies have shown that patients with severesepsis or septic shock showed an increased likelihood of death of 7.6%for every hour in which antibiotic therapy is not applied. Survivalrates are also significantly reduced when antibiotics are not appliedwithin the first 6 hours of identifying hypotension. Survival rates willbe significantly increased if diagnosis times are reduced.

Culturing microorganisms from blood samples (Gram staining) remains thegold standard in detection of the microbiological cause of sepsis. Thismethod is however subject to significant disadvantages, in particular,due to the large time difference between taking a blood sample andproviding the results. It is not uncommon that 24 to 72 hours passbetween taken a sample and providing diagnostic information. Within thistime, broad band, untargeted antibiotic therapies are often introduced.This may lead to some success in treating the disease but is related tosignificant disadvantages with respect to the development of antibioticresistant microorganisms.

Microarray and multiplex PCR approaches have been disclosed in the art,which are typically defined by extremely large numbers of probes orprimers required for application of such methods (leading to significantcost and effort), a limited pool of target pathogens capable of beingdetected (such as only a limited sub-group of bacterial pathogens, orthe absence of fungal pathogens), or a lack of discrimination betweengram-negative and gram-positive bacterial pathogens, which providessub-standard information for appropriate antibiotic therapies (US2009286691 and US 201 1 151453). Methods for discriminatinggram-positive and gram-negative bacteria have been disclosed in the art(US 20080118923 A1), in addition to the combined analysis of 16S and 18Ssequences of bacteria and fungi (US 20090061446). Such methods, althoughpotentially useful in clinical diagnostics, have never been applied insepsis analytics and employ large numbers of primers in eithermicroarray or very complex multiplex reactions, representing asignificant technical and financial challenge for clinical diagnosticlaboratories.

Multiplex RT-PCR approaches have been described in which a number ofcommonly occurring human pathogens are potentially detected. One exampleof such a multiplex PCR method is described in Gosiewski et al (BMCMicrobiology 2014, 14:144) and U.S. Publication No. 2015/0232916, whichdiscloses a nested PCR approach for detecting gram-positive andgram-negative bacteria, yeast, fungi and filamentous fungi from bloodsamples. A nested polymerase chain reaction involves two sets ofprimers, used in two successive runs of polymerase chain reaction, thesecond set intended to amplify a secondary target within the first runproduct. Nested PCR is applied in order to reduce nonspecific binding inproducts due to the amplification of unexpected primer binding sites, asit is unlikely that any of the unwanted PCR products contain bindingsites for both the new primers in the second PCR run, ensuring theproduct from the second PCR has little contamination from unwantedproducts of primer dimers, hairpins, and alternative primer targetsequences. Despite potentially reducing background signal, the PCRmethod described in Gosiewski and U.S. Publication No. 2015/0232916 arerelatively complex and require two cycling reactions, essentiallydoubling the time, effort and reagents required for the analysis.

Other methods have employed the amplification of a number of PCRproducts from bacterial and fungal pathogens using sequence-specificoligonucleotides together with sequence-unspecific dyes, andsubsequently, a melting curve analysis to differentiate between thevarious products (Horvath et al, BMC Microbiology 2013, 13:300). Themethod disclosed therein is however limited by a number of disadvantagesknown to occur with melting curve analyses.

Other methods have employed the amplification of a number of PCRproducts from bacterial and fungal pathogens using non-sequence-specificoligonucleotides together with sequence-specific probes to differentiatebetween the various products as described in EP 3172337. In such cases,only a broadband antibiotic therapeutic approach is possible, which may,in fact, be poorly suited for the particular pathogen.

Electrochemical detection techniques have higher detection sensitivitythan conventional luminescence techniques (e.g., fluorescence andphosphorescence) due to higher signal-to-noise ratios. Because of theirsensitivity and ability to accurately measures low-concentrations ofnucleic acids, electrochemical detection techniques are able todifferentiate between pathogenic species representing a significanttechnological improvement over the prior art. But, because of theirsensitivity, false positive detection rates are high. Indeed, whereorganisms are cultured, the growth media often contains non-viableorganisms or DNA/nucleic acids, which would not affect culture, butcould produce false positives in PCR. If a system is designed uniformlyfor increased sensitivity to detect low titers pathogens, frequent falsepositive results may occur from background organisms or DNA/nucleicacids. Alternatively, if system sensitivity is reduced to avoidbackground organism detection, low titer organisms may be missed,resulting in false negative detection.

Further, when blood or other bodily fluids are obtained from a subjectthey may be contaminated by skin cells, bacteria, fungi, viruses,phages, their respective nucleic acids (including RNA and DNA) and/orother undesirable molecules, or disinfectants. Antiseptics are crucialfor the practice of medicine; however, currently used antiseptics have asignificant failure rate which results in substantial additional medicalcosts. Antiseptics are commonly used prior to routine phlebotomy, inpreparation for minor and major invasive procedures, and as part ofroutine infection control hand-washing practices. The failure ofantiseptics often result in erroneous diagnostic tests. For example, ithas been estimated that a single false positive blood culture (i.e.,where the culture indicates that the blood has been infected withbacteria, although the blood was contaminated during the blood draw)done on blood drawn from a patient at a hospital costs the patient anadditional S2000 to S4,200 in unnecessary medication, additional followup testing, and increased length of stay. (Bates, 1991).

Thus, there is a need in the art to provide methods which canselectively detect pathogenic organisms of interest. In particular,there is a need in the art for a method which enables the discriminationbetween a systemic infection and a false positive signal due to bloodmatrix bottle contamination. There is also a need in the art to identifywhen a blood culture is contaminated during blood draw.

BRIEF SUMMARY OF THE INVENTION

The ability to detect infection is hampered by background contaminationpresent in blood culture bottles, such as are used in gram staining, acommon first step in any clinical pathogen diagnosis. The inventiondisclosed herein can not only differentiate between backgroundcontamination (from any blood culture matrix bottle) and clinicallyrelevant infection but can also differentiate between gram-positivebacterial infection, gram-negative bacterial infection, fungal infectionand can identify antibiotic resistance genes. Even more importantly, theinvention can identify the contaminating pathogen and contaminatingco-infection (if present) by its species. Because prior art methodsfailed to recognize background contamination as an issue or cannotdiscriminate by species the infecting pathogen and co-infectingpathogen, the invention allows for better antimicrobial stewardship andimproved patient care outcomes.

Disclosed herein are in vitro methods (or systems or devices) for thedetection and/or identification of a human pathogen and/or geneticmaterial thereof comprising subjecting a sample suspected of comprisinga human pathogen and/or genetic material thereof to a single multiplexpolymerase chain reaction (PCR), wherein said method (or system)comprises amplification of PCR products that enable discriminationbetween contaminating pathogen and/or genetic material present in thesample and infectious pathogen and/or genetic material present in thesample. In particular, the inventive method allows for a singleamplification step and single detection step for the detection of anactual pathogenic infection and not a putative contamination. Theinventive method does not require a purification step prior toamplification or detection. The inventive method does not requiredetermining whether amplification has occurred prior to detection. Whenpurification is needed or a determination as to whether amplificationhas occurred prior to detection requires human action and such systemscannot be fully automated like the invention disclosure herein. Theinventive method does not require dilution of the PCR sample. Theinventive method does not require additional testing or analysis todifferentiate signaling due to contamination versus real pathogenicinfections.

The methods (or system or devices) can further discriminate betweengram-positive bacterial, gram-negative bacterial and fungal pathogens ifpresent in said sample as well as identify antimicrobial resistancegenes. An infection can be identified by its species, and a fungalco-infection can be identified by its genus whereas a bacterialco-infection of a different type than the infection (i.e. infection isGP and co infection is GN or vice versa) can be identified by itscategory (gram-positive or negative). If the infection and co-infectionare of the same type (i.e., both gram-positive or both gram-negative),the systems and methods can identify the species of the co-infection viaa single PCR run. If the infection and co-infection are of differenttypes (i.e., the infection is gram-positive and the co-infection isgram-negative or fungal) the systems and methods can identify thespecies of the co-infection via a second single PCR run.

The methods (or system or devices) can further discriminate betweenbackground contamination and de-escalation targets. Backgroundcontamination from blood culture bottles is not detected by the methods(or system or devices) but organisms associated with possiblecontamination by blood draw such as Propionibacterium acnes,Staphylococcus epidermidis, Micrococcus, Lactobacillus orCorynebacterium (so called de-escalation targets) are identified. Themethods (or system or devices) can further discriminate between (1)background contamination, (2) de-escalation targets and (3) clinicallyrelevant gram-positive bacterial, gram-negative bacterial or fungalpathogens if present in the sample as well as identify antimicrobialresistance genes.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1: Shows a schematic of a hybridization complex.

FIG. 2: Shows a schematic of the bi-directional LIS to automate andaccelerate order entry and results reporting.

FIG. 3: Shows a schematic of “Order-to-report.” The clock time stampsare commonly documented in hospital and laboratory information systems.

FIG. 4: BCID-GP, High false positive signals for Enterococcus faecalis,Pan-candida, and Pan-GN in bottle matrix with no blood or bacterialtargets.

FIGS. 5A-5D: False positives detected for Enterococcus faecalis (5A),Staph 16s (5B); Enterococcus (genus) (5C); and Pan-Candida (5D), weredetected when negative blood culture matrices (no blood or bacterialtargets) were tested on a BCID-GP cartridge.

FIGS. 6A-6B: FIG. 6A shows Enterococcus faecalis contamination signalsare reduced but not eliminated with 35 cycles and FIG. 6B shows that at35 cycles the S. pombe internal control is still detected.

FIGS. 7A-7D: False positive signal from blood culture bottles iseliminated using a 30-cycle PCR. FIGS. 7A and 7B show that only thepositive controls were detected. FIGS. 7C and 7D show that detection ispossible (although weak) at 1×LOD (1×105 CFU/mL) Enterococcus faecalisrun on sLRMs using a 30-cycle PCR.

FIG. 8: BCID-GP, Background P. acnes signals with 30, 35, and 40-cyclePCR. False Positives are detected with 40 and 35 cycles when BDIC-GPcartridges are spotted with P. acnes primers but eliminated with 30cycle PCR.

FIGS. 9A-9B: BCID-GP, P. acnes detection at 1×LOD with 30-cycle PCR.When 30 PCR cycles are used, P. acnes is detected at 1×LOD (1×106CFU/mL) (9A for P. acnes strain ATCC11827 and 9B for P. acnes strainATCC6919).

FIGS. 10A-10B: BCID-GP, Strep spp. assay performance with 30-cycle PCR.FIG. 10A shows that when 30 PCR cycles are used, six representativeStreptococcus species identified by their identigifcation number aredetected at 1×LOD (1×106 CFU/mL). FIG. 10B corrolates the bacterialspecies name and identification number.

FIG. 11: BCID-GP cartridge with 1×LOD Streptococcus spp. When primerconcentration is increased, three P. acnes strains and two Streptococcusspecies are detected at 30 PCR cycles and 1×LOD (1×106 CFU/mL).

FIG. 12: Contamination of NTC sLRMs with Streptococcus spp. and P. acnesprimers and 30 cycles of PCR. No Streptococcus spp or P. acnes signalswere detected with increased primer concentrations and 30 PCR cycles innegative blood culture matrices.

FIG. 13: BCID-GP, PCR cycles are reduced from 40 to 37. When PCR cyclesare reduced from 40 to 37, most blood matrix contamination iseliminated.

FIG. 14: BCID-GP Multiplex primer pool and PCR cycles.

FIG. 15: PCB with GP Reagent & 8 PCR Drop Locations: a schematic of theBCID-GP cartridge sub-assembly layout. Reservoirs, R1, R2, R3, R4, andR5 are part of the top plate. R1 typically includes PCR enzyme (Taq) andbuffer; R2 typically includes exonuclease; R3 typically includesreconstitution buffer used to wet reagents and rehydrate PCR reagents;R4 typically includes is a waste manipulation zone or is empty for dropmanipulation; and R5 is typically where the sample comes out of the LRMafter extraction. “Sample” designates where the sample is loaded fromthe LRM. “PCR enzyme” means Taq and “PCR buffer” is a buffer; the bufferand PCR enzyme are shown where they are spotted on the top plate. “Exo”is exonuclease and is shown where it is spotted on the top plate. 1, 2,3, 4, 5, 6, 7, 8 displayed vertically next to heater 3 are the multiplexprimer pools; once the drop has the primers they go into PCR lane 1, 2,3 or 4 as shown and cycled (35 or 30) as shown. 1, 2, 3, 4, 5, 6, 7, 8displayed vertically on Heater 1 or 3 depict where each PCR drop ismoved. SPCA means signal probe cocktail which is where the amplicon ismixed with the signal probe. Detection zones A, B, C and D are wheredetection takes place. The drops 1, 2, 3, 4, 5, 6, 7, 8 are moved intothe detection zone as shown. The gold plated electrode is depicted assmall round circles in the detection zone. The RTD temperature setpoints are shown in degrees Celsius.

FIGS. 16A-16B: Shows false positive signal for Proteus mirabilis (FIG.16A) and Proteus spp (FIG. 16B) in negative blood culture matricescycled 40 times (a variety of blood culture bottles are shown).

FIG. 17: BCID-GN, Negative bottles run with reduced cycling showed nofalse positives. When negative blood culture matrices were cycled 35 or30 times (cycling as indicated in FIG. 17) no false positives weredetected.

FIG. 18: BCID-GN Multiplex primer pool and PCR cycles. The boldedorganisms are the genus calls in the detection report and the non-boldedorganisms are species calls on the detection report.

FIG. 19: A schematic of the BCID-GN cartridge sub-assembly layout.Reservoirs, R1, R2, R3, R4, and R5 are part of the top plate. R1typically includes PCR enzyme (Taq) and buffer; R2 typically includesexonuclease; R3 typically includes reconstitution buffer used to wetreagents and rehydrate PCR reagents; R4 typically includes is a wastemanipulation zone or is empty for drop manipulation; and R5 is typicallywhere the sample comes out of the LRM after extraction. “Sample”designates where the sample is loaded from the LRM. “PCR buffer” is abuffer; the buffer and taq are shown where they are spotted on the topplate. “Exo” is exonuclease and is shown where it is spotted on the topplate. MP1, MP2, MP3, MP4, MP5, MP6, MP7, MP8 displayed vertically nextto heater 3 are the multiplex primer pools; 1, 2, 3, 4, 5, 6, 7, 8displayed vertically on Heater 1 or 3 depict where each PCR drop ismoved. Once the drop has the primers they go into PCR lane 1, 2, 3 or 4and cycled (35 or 30) as shown in the key below. SPC means signal probecocktail which is where the amplicon is mixed with the signal probe.Detection zones A, B, C and D is where detection takes place. The drops1, 2, 3, 4, 5, 6, 7, 8 are moved into the detection zone as shown. Thegold plated electrode is depicted as small round circles in thedetection zone. The RTD temperature set points are shown in degreesCelsius.

FIGS. 20A-20B: BCID-FP, Detuning Eliminates False Positives. The signalsobtained before and after detuning, for Rhodotorula (FIG. 20A) andTrichosporon (FIG. 20B) are shown.

FIG. 21: The BCID-FP Multiplex primer pool and PCR cycles.

FIG. 22: BCID-FP, PCB with Fungal Reagent & PCR Drop Locations. Aschematic of the BCID-FP cartridge sub-assembly layout is shown.Reservoirs, R1, R2, R3, R4, and R5 are part of the top plate. R1typically includes PCR enzyme (Taq) and buffer; R2 typically includesexonuclease; R3 typically includes reconstitution buffer used to wetreagents and rehydrate PCR reagents; R4 typically includes is a wastemanipulation zone or is empty for drop manipulation; and R5 is typicallywhere the sample comes out of the LRM after extraction. “Sample”designates where the sample is loaded from the LRM. “PCR buffer” is abuffer; the buffer and taq are shown where they are spotted on the topplate. “Exo” is exonuclease and is shown where it is spotted on the topplate. PM1, PM 2, PM3 and PM4 displayed vertically next to heater 3 arethe multiplex primer pools 1-4; MP1, MP2, MP3 and MP4 means PCR primermixes; 1, 2, 3, 4 displayed vertically on Heater 3 depict where each PCRdrop is moved. Once the drop has the primers they go into PCR lane 1, 2,3 or 4 and cycled 40 times. SPC means signal probe cocktail which iswhere the amplicon is mixed with the signal probe. Detection zones A, B,C and D is where detection takes place. The drops 1, 2, 3, and 4 aremoved into the detection zone as shown. The gold plated electrode isdepicted as small round circles in the detection zone. cDT-8 and cDT-9are the controls used. cDT-8 uses a ferrocene derivative QW56. cDT-9uses a ferrocene derivative QW56. The RTD temperature set points areshown in degrees Celsius.

FIG. 23: A notice showing that if the BCID Panel is used to testBacT/ALERT SN bottles, positives for Pseudomonas aeruginosa andEnterococcus should be reconfirmed by another method prior to reportingthe test results.

DEFINITIONS

“Target nucleic acid,” or “analyte of interest”, or “target molecule” or“human pathogen nucleic acid”, include genes, portions of genes,regulatory sequences of genes, mRNAs, rRNAs, tRNAs, siRNAs, cDNA and maybe single stranded, double stranded or triple stranded. As discussedherein, target nucleic acids are DNA from human pathogens, and arenaturally occurring nucleic acids, as contrasted to the nucleic acids ofcapture probes and signal probes, which may include non-naturallyoccurring components. Some nucleic acid targets have polymorphisms,single nucleotide polymorphisms, deletions and alternate splicesequences, such as allelic variants. Multiple target domains may existin a single molecule, for example, a target nucleic acid may have afirst target domain that binds the capture probe and a second targetdomain that binds a signal probe, and/or distinct primer bindingsequences. Target nucleic acids are not generally provided with thecartridge as manufactured, but are contained in the liquid sample to beassayed; in contrast, “control analytes” or “control nucleic acids” aretypically provided with the cartridge or are routinely present in asample of a particular type and are assayed in order to ensure properperformance of the assay. Spiked samples may be used in certain qualitycontrol testing and for calibration, as is well known in the art. Thetarget analyte is also referred to as “clinically relevantamplification” or “systemic infection” or “pathogen of interest” and isdistinguished from, for example, contamination.

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include bloodproducts, such as plasma, serum and the like. In contrast with somecommercial systems that require some off chip handling of the sample,generally including sample extraction (cell lysis, for example), andsample preparation prior to detection. Thus, in accordance with aspectsof the current system, a sample is loaded onto a BCID cartridge and thetarget analyte is extracted, amplified as necessary (for example, whenthe target analyte is a nucleic acid using polymerase chain reaction(PCR) techniques, although isothermal amplification methods can beutilized as well), and then detected using electrochemical detection,all on a microfluidic platform, generally referred to herein as a“multiplex cartridge” or a “fluid sample processing cartridge.” The BCIDcartridge utilizes a sample preparation module as further described andshown in FIG. 15 of U.S. Pat. No. 9,598,722 (which is hereinincorporated by reference in its entirty). In many embodiments, e.g. forthe detection of human pathogens, the sample is a blood sample that istreated as outlined herein. Environmental samples include environmentalmaterial such as surface matter, soil, water, crystals and industrialsamples. Such examples are not, however, to be construed as limiting thesample types applicable to the present technology.

By “nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, for example in the creation of signalprobes and sometimes capture probes, nucleic acid analogs are includedthat may have alternate backbones, comprising, for example,phosphoramides, phosphorothioates, phosphorodithioates,Omethylphophoroamidite linkages and peptide nucleic acid backbones andlinkages, as well as those with positive backbones, non-ionic backbonesnonribose backbones, including those containing one or more carbocyclicsugars are also included within the definition of nucleic acids. Thesemodifications of the ribosephosphate backbone may be done to facilitatethe addition of electron transfer moieties, or to increase the stabilityand half-life of such molecules in physiological environments.

The term “detection system” as used herein refers to a method thatenables visualization of PCR-amplified nucleic acid products. Examplesof suitable detection systems include systems that depend on detectionof color, radioactivity, fluorescence, chemiluminescence orelectrochemical signals, with the latter finding particular use in thepresent invention.

The term “contamination” or “contaminant” or “background contamination”or “contaminating pathogen and/or genetic material” or “unwantedcontamination” as used herein refers to nucleic acids in the samplewhich are not a part of the nucleic acid population that is beingtargeted for amplification. For example, nucleic acids found in theblood culture matrix.

The term “de-escalation targets” means Propionibacterium acnes,Staphylococcus epidermidis, Micrococcus, Lactobacillus orCorynebacterium. An object of the invention is to distinguish betweenunwanted contamination (from blood culture bottles) and de-escalationtargets (which may be present as a contamination from blood draw) butwhich may be a clinically relevant infection. An object of the inventionis to distinguish between unwanted contamination, de-escalation targets,clinically relevant pathogens and determinants of antimicrobialresistance.

The term “infection” means the invasion of a host organism's body byanother organism or entity (pathogen), for example, a fungi or bacteria.The meaning of the term “co-infection” as used herein means “doubleinfection,” “multiple infection,” or “serial infection” and is used todenote simultaneous infection with two or more infections/pathogens.

The term “determinants of antimicrobial resistance” relates to a generesponsible for the development of resistance in the bacteria whichactively counteracts the effect of an antibiotic. Particularly, geneticdeterminants of resistance to methicillin (mecA and mecC) and vancomycin(vanA and vanB) are envisaged. Genes associated with geneticdeterminants of resistance such as CTX-M, NDM, IMP, OXA, KPC, VIM areenvisaged.

For some nucleic acid detection systems, the target sequence isgenerally amplified, and during amplification, a label is added. Thecompositions of the invention may additionally contain one or morelabels at any position. By “label” herein is meant an element (e.g. anisotope) or chemical compound that is attached to enable the detectionof the compound. Preferred labels are radioactive isotopic labels, andcolored or fluorescent dyes. The labels may be incorporated into thecompound at any position. In addition, the compositions of the inventionmay also contain other moieties such as cross-linking agents tofacilitate cross-linking of the target-probe complex. See for example,Lukhtanov et al., Nucl. Acids. Res. 24(4):683 (1996) and Tabone et al.,Biochem. 33:375 (1994), both of which are expressly incorporated byreference.

The electrochemical detection system used herein uses a separate singalprobe or label probe having an electron transfer moiety (ETM). That is,one portion of the label probe directly or indirectly binds to thetarget analyte, and one portion comprises a recruitment linkercomprising covalently attached ETMs. In some systems, these may be thesame. In an embodiment, the ETM is responsive to an input waveform. Inan embodiment, the ETM is a metallocene. In an embodiment, themetallocene is a ferrocene. In an embodiment, the ferrocene is aferrocene derivative. Preferred ferrocene derivatives can be N6 (FIG. 1Das shown in U.S. application Ser. No. 14/218,615), QW56 (FIG. 2A asshown in U.S. application Ser. No. 14/218,615), and QW80 (FIG. 2B asshown in U.S. application Ser. No. 14/218,615).

The expression “electrochemical system” or “electrochemical detectionsystem” or “automated nucleic acid testing system” refers to a systemthat determines the presence and/or quantity of a redox analyte throughmeasurements of electrical signal in a solution between a workingelectrode and a counter electrode, such as induced by a redox reactionor electrical potential from the release or absorption of ions. Theredox reaction refers to the loss of electrons (oxidation) or gain ofelectrons (reduction) that a material undergoes during electricalstimulation such as applying a potential. Redox reactions take place atthe working electrode, and which, for chemical detection, is typicallyconstructed from an inert material such as platinum or carbon. Thepotential of the working electrode is measured against a referenceelectrode, which is typically a stable, well-behaved electrochemicalhalf-cell such as silver/silver chloride. The electrochemical system canbe used to support many different techniques for determining thepresence and concentration of the target biomolecules including, but notlimited to, various types of voltammetry, amperometry, potentiometry,coulometry, conductometry, and conductimetry such as AC voltammetry,differential pulse voltammetry, square wave voltammetry, electrochemicalimpedance spectroscopy, anodic stripping voltammetry, cyclicvoltammetry, and fast scan cyclic voltammetry. The electrochemicalsystem may further include one or more negative control electrode and apositive control electrode. In the context of the invention, a singleelectrochemical system may be used to detect and quantify more than onetype of target analyte. The use of electrochemical systems is describedin more detail in U.S. Pat. Nos. 9,557,295, 8,501,921, 6,600,026,6,740,518 and U.S. application Ser. No. 14/538,506 which are hereinincorporated by reference in their entirety.

The term “pathogen” or “human pathogen” as used herein refers to anorganism (bacteria or fungi) that may affect the health status of thehost, if that host is infected by that organism. A large number of humanpathogens are outlined in the Tables, Examples and Lists herein.Included within the definition of human pathogen is the geneticmaterial, usually DNA, that is contained within the pathogenic organism.In addition, as will be appreciated by those in the art, included withinthe definition of the genetic material of a pathogen are amplicons thatresult from amplification reactions such as the PCR reactions describedherein.

The term “analyzing the presence of a pathogen” is used to describe amethod to determine the presence or absence of a pathogen. The systemsand methods disclosed herein do not require additional analysis todiscriminate between background signaling due to contamination effectsand real pathogenic infections and thus enable a decision on whether toapply a selective antibiotic therapy.

The term “thresholding” or “threshold signal” or the like refers to aset signal level below which the reported call is “not detected,” abovewhich the reported call is “detected.”

The term “PCR” means “polymerase chain reaction.” PCR is a techniqueused in molecular biology to amplify a single copy or a few copies of asegment of DNA across several orders of magnitude, generating thousandsto millions of copies of a particular DNA sequence. PCR reagentsgenerally include pairs of primers, dNTPs and a DNA polyermase.

The term “single reaction” or “single run” or “single multiplex PCR” or“single PCR” or “single nucleic acid amplification reaction” or “singleamplification” or the like in this context refers to a standard PCRoperating program. A single PCR run encompasses non-uniform PCR cycling(also referred to as heterogeneous PCR cycling, non-harmonized, uneven,unsymmetrical, mismatched PCR cycling and the like) in a singlecartridge, i.e., some samples being cycled 30 times while others arecycled 35 times but not two sequential PCR runs such as with nested PCR.If heterogeneous single run PCR cycling were not utilized, there wouldbe either a risk of false positives for the organisms that tend to havehigh contamination concentrations (such as Bacillus) or a risk of falsenegatives for organisms that tend to have slower growth in culture andtherefore fewer copies of target sequence in the sample (such as E.Coli), or both if a compromise cycle were chosen. Using heterogeneoussingle run PCR cycles for different organisms improves the overallaccuracy of the assay. Herein, the standard PCR operating programcomprises a series of repeated temperature changes, called cycles, witheach cycle consisting of 2 discrete temperature steps, referred to asdenaturation and annealing/extension steps. The cycling is preceded by asingle temperature step (hot start) at a high temperature (>90° C.) forenzyme activation.

“Nucleotide” means a building block of DNA or RNA, consisting of onenitrogenous base, one phosphate molecule, and one sugar molecule(deoxyribose in DNA, ribose in RNA).

“Oligonucleotide” means a short string of nucleotides. Oligonucleotidesare often used as probes to find a matching sequence of DNA or RNA andcan be labeled with a variety of labels, such as radioisotopes andfluorescent and chemiluminescent moieties and ferrocene labels.

“Primer” means a short strand of oligonucleotides complementary to aspecific target sequence of DNA, which is used to prime DNA synthesis.Some primer pools contain species-specific primers. Suchspecies-specific primer pairs hybridize in the assay to a target nucleicacid sequence of only one of said target species (gram-positivebacterial, gram-negative bacterial or fungal). Some primer pools containgenus-specific primers. Each double stranded amplicon contains ablocking moiety (phosphorylation on one strand) so that exonucleaseactivity is blocked, thereby inhibiting digestion of the blocked strandand promoting digestion of the unblocked strand. Exonucleases areenzymes that work by cleaving nucleotides one at a time from the end(exo) of a polynucleotide chain. A hydrolyzing reaction that breaksphosphodiester bonds at either the 3′ or the 5′ end occurs.

“Uniplex” means a PCR-based assay utilizing a single set of primers ineach reaction that amplifies a single pathogen specific nucleic acidsequence

“Multiplex” means a PCR-based assay utilizing multiple primer sets in asingle reaction, where each primer can amplify a single pathogenspecific nucleic acid sequence.

“End point PCR” means one multiplexed PCR method for amplification andend point detection (i.e. after the log phase).

“Real-time PCR” or “Q-PCT” refers to a homogenous PCR assay that permitscontinuous fluorescent monitoring of the kinetic progress of theamplification reaction. Methods of conducting real-time PCR are wellknown in the art and a number of systems are available commercially (seee.g. Higucho et al., “Kinetic PCR Analysis: Real-time Monitoring of DNAAmplification Reactions,” Bio/Technology 11:1026-1030 (1993))

The term “capture probe” refers to the nucleic acid sequence, specificto the individual pathogen that is immobilized on an inert matrix. Whena capture probe is combined with other capture probes for simultaneousdetection of multiple pathogens, the specificity of the capture probeshould not be substantially affected by the presence of other captureprobes, i.e., it still hybridizes to the target pathogens nucleic acid.Preferably, a capture probe selected for one pathogen does not hybridizeto a nucleic acid from another pathogen. Capture probes generallyhybridize to a first target domain of an amplicon of a human pathogen asoutlined herein.

The term “signal probe” refers to the nucleic acid sequence, specific tothe individual pathogen that is not immobilized on an inert matrix.Signal probes generally hybridize to a second target domain of anamplicon of a human pathogen as outlined herein, and they are generallylabeled. Signaling probes in some embodiments are labeled with differentlabels that enable simultaneous use and differentiation between each ofthe labels. However, in the BCID-GP and GN panels disclosed thesignaling probes are not labelled with different labels such that whenthe signaling probe binds, “pan-candida detection” is reported not thespecific candida species detected (Candida albicans, Candida glabrata,Candida krusei, Candida parapsilosis).

By “pan-assay” or “pan-target” in the context of the invention herein ismeant an assay that detects if a marker such as a gene is present in thesample and is reflective of the presence of a pathogen category such asa gram-positive bacteria, gram-negative bacteria or fungi. Pan-assaysare characterized by the fact that they reflect the possibility of thepresence of more than one pathogen in the sample. Thus, pan-assays arenot specific for a single pathogen being present in the sample, but arespecific for a pathogen type such as gram-positive, gram-negative orfungi in the sample.

“Hybridization” refers to the process of joining two complementarystrands of nucleic acid to form a double-stranded molecule; morespecifically mentioned here is hybridization between the ‘probe (captureor signal)’ and the ‘target’ nucleic acid sequences. In manyembodiments, a “hybridization complex” comprises three nucleic acids: atarget nucleic acid, a signal probe hybridized to a first target domainof the target nucleic acid and a capture probe hybridized to a secondtarget domain of the target nucleic acid.

As used herein, the term “cartridge” or “consumable” is a Self-containedcartridge/consumable that includes the necessary components to perform asingle BCID Panel test. A “cartridge” or “consumable” is a cartridge forperforming assays in a closed sample preparation and reaction system asdescribed in U.S. Pat. No. 9,598,722 which is herein incorporated byreference in its entirety. The invention provides cartridges comprisingseveral components, including a biochip cartridge, a top plate, a liquidreagent module (LRM), and a housing that keeps the components together.The biochip cartage comprises a bottom substrate, a sample preparationzone, reagent zone, Sample Manipulation Zone, Amplification Zone,Detection Zones as further described in U.S. Patent Publication no.2015/0323555 and U.S. Pat. No. 9,598,722 which are herein incorporatedby reference in their entireties. Specifically, in the embodiments fordetecting nucleic acid targets, the substrate comprises one or moreamplification pathways/zones. The top plate is spotted with reagents andprimers. During the spotting process, phenol red is added to thereagents and primers so that spotting can be visualized. The LRMincludes fluid filled blisters, as generally depicted in FIG. 1 fromU.S. Patent application publication no. 2014/0194305 which is hereinincorporated by reference in its entirety. For example, lysis buffer(which in some cases can be water for hypotonic lysis, or can be acommercially available lysis buffer, such as those containing chiatropicsalts such as guanidinium salts, and or high/low pH, and/or surfactantssuch as sodium dodecyl sulfate (SDS), Polysorbate 20, Triton-X, etc. iscontained within a blister that is activated to add lysis buffer to thesample. These buffers and in particular Polysorbate 20 (such as Tween®20) can be washed or they can remain in the sample upon amplification.The top plate may include a PDOT (or PEDOT) coating. PEDOT:PSS orpoly(3,4-ethylenedioxythiophene) polystyrene sulfonate is a polymermixture of two ionomers. One component in this mixture is made up ofsodium polystyrene sulfonate which is a sulfonated polystyrene. Part ofthe sulfonyl groups are deprotonated and carry a negative charge. Theother component poly(3,4-ethylenedioxythiophene) or PEDOT is aconjugated polymer and carries positive charges and is based onpolythiophene. Together the charged macromolecules form a macromolecularsalt. The top plate may be coated with Teflon®, Cytop®, or Fluoropel®,preferably Cytop®. Cytop® is an amorphous fluoropolymer with highoptical transparency and excellent chemical, thermal, electrical andsurface properties. As used herein, the term “cartridge sub-assembly”means the bottom plate and top plate together.

As used herein, the term BCID-GP means Blood CultureIdentification—Gram-Positive Panel. The BCID-GP panel includes all ofthe oligonucleotides and reagents for carrying out a nucleic acidamplification reaction for the targets listed in FIG. 14 as well as thecapture and signal probes to form the hybridization complex necessary todetect the targets listed in FIG. 14. Specifically, phenol red isincluded in the reagents and primer mix pools as a visual tool to ensurethe top plates are properly spotted.

As used herein, the term BCID-GN means Blood CultureIdentification—Gram-Negative Panel. The BCID-GN panel includes all ofthe oligonucleotides and reagents for carrying out a nucleic acidamplification reaction for the targets listed in FIG. 18 as well as thecapture and signal probes to form the hybridization complex necessary todetect the targets listed in FIG. 18. Specifically, phenol red isincluded in the reagents and primer mix pools as a visual tool to ensurethe top plates are properly spotted.

As used herein, the term BCID-FP means Blood CultureIdentification—Fungal Panel. The BCID-FP panel includes all of theoligonucleotides and reagents for carrying out a nucleic acidamplification reaction for the targets listed in FIG. 21 as well as thecapture and signal probes to form the hybridization complex necessary todetect the targets listed in FIG. 21. Specifically, phenol red isincluded in the reagents and primer mix pools as a visual tool to ensurethe top plates are properly spotted.

As used herein, the term “BCID-GP cartridge” or “BCID-GN cartridge” or“BCID-FP cartridge” means a cartridge for performing gram-positive,gram-negative, or fungal assays respectively in a closed samplepreparation and reaction system as described in U.S. Pat. No. 9,598,722which is herein incorporated by reference in its entirety.

As used herein, the term “about” means encompassing plus or minus 10%.For example, about 90% refers to a range encompassing between 81% and99% nucleotides. As used herein, the term “about” is synonymous with theterm approximately.

Unless otherwise indicated or the context suggests otherwise, as usedherein, “a” or “an” means “at least one” or “one or more.”

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

The term “amplifying” or “amplification” in the context of nucleic acidsrefers to the production of multiple copies of a polynucleotide(generally referred to herein as “amplicons”), or a portion of thepolynucleotide, typically starting from a small amount of thepolynucleotide or a single polynucleotide molecule, where theamplification products or amplicons are generally detectable. Detectionin the system ranges, for example, on the low end C. Kefyr is 200 CFU/mLwithout false positives due to contaminants. For fungal the upperdetection limit for organisms is 1×10⁵. For gram-negative bacteria thedetection limit for organisms ranges from 1×10⁵ to 1×10⁷ without falsepositives due to contaminants. For gram-positive bacteria the detectionlimit for organisms ranges from 1×10⁵ to 1×10⁸ without false positivesdue to contaminants.

Amplification of polynucleotides encompasses a variety of chemical andenzymatic processes. The generation of multiple nucleic acid copies fromone or a few copies of a target or template nucleic acid molecule duringa polymerase chain reaction (PCR) or a ligase chain reaction (LCR) areforms of amplification.

The term “detect”, “detecting” or “detection” refers to an act ofdetermining the existence or presence of one or more targets (e.g.,microorganism nucleic acids, amplicons, etc.) in a sample. As usedherein, target detection occurs when the amplicon forms a hybridizationcomplex with the complimentary signal and capture probe.

Amplicon—double-stranded nucleic acid product of PCR. Generally, theamplicon comprises a length that is compatible with electrochemicaldetection which is typically less than 300 base pairs although manyamplicons used herein are less than 150; indeed some amplicons used inthe system are less than 100 base pairs. Preferably the amplicon is lessthan 300 base pairs, 200 base pairs, 150 base pairs, 100 base pairs, or75 base pairs. Generally, the goal is to make a short amplicon becauseit is more efficient for exonuclease to make it single strand and alsorequires shorter amplification times.

“Bay” or “instrument bay” or “cartridge bay”—Stand-alone processing unitwhich runs a consumable. Bays as used herein are further described inU.S. patent application Ser. No. 14/062,860, U.S. Patent Publication no.2015/0323555 and U.S. Pat. No. 9,598,722 which are herein incorporatedby reference in their entireties.

Exonuclease digestion—enzyme-driven process digesting double-strandednucleic acid to single-stranded nucleic acid fragments. Exonucleaseactivity is blocked by phosphorylating one strand, thereby inhibitingdigestion of the blocked strand and promoting digestion of the unblockedstrand.

RTD—Temperature set point that is controlled by a feedback loop from theresistance temperature detectors (RTDs) to the Thermistor on the bay.

sLRM—“simulated liquid reagent module”, a blood culture sample that ismanually prepared on the bench to mimic processing on an automatedinstrument.

“Open bay” means an bay lacking the top plate bay component so onlycartridge-related functions can be performed

NTC sLRM=No Template Control sLRM is a sLRM prepared without positiveblood culture or bacterial targets

NTC—No template control

DETAILED DESCRIPTION OF THE INVENTION

While aspects of the subject matter of the present disclosure may beembodied in a variety of forms, the following description andaccompanying drawings are merely intended to disclose some of theseforms as specific examples of the subject matter. Accordingly, thesubject matter of this disclosure is not intended to be limited to theforms or embodiments so described and illustrated.

Unless defined otherwise, all terms of art, notations and othertechnical terms or terminology used herein have the same meaning as iscommonly understood by one of ordinary skill in the art to which thisdisclosure belongs. All patents, applications, published applicationsand other publications referred to herein are incorporated by referencein their entirety. If a definition set forth in this section is contraryto or otherwise inconsistent with a definition set forth in the patents,applications, published applications, and other publications that areherein incorporated by reference, the definition set forth in thissection prevails over the definition that is incorporated herein byreference.

I. Introduction

The present disclosure relates to methods and systems for distinguishingbetween background contamination and clinically relevant infection. Asnoted above, the ability to detect pathogen infections in humans ishampered by background contamination present in the blood culturebottles used during gram-staining, the first step in many diagnoses. Thepresent invention can distinguish between background contamination andthe pathogen, including situations where the patient has more than oneinfection (e.g. a primary infection and a co-infection). The presentinvention can further identify the presence of de-escalation targets ina sample wherein the de-escalation target is Propionibacterium acnes,Staphylococcus epidermidis, Micrococcus, Lactobacillus orCorynebacterium.

The methods and systems can further distinguish between gram-positive,gram-negative and fungal infection(s). The methods and systems canfurther detect and identify antimicrobial resistance genes. If theinfection is gram-positive or gram-negative the species of the infectioncan be identified. If a co-infection is present, and is of the samecategory as the infection (both gram positive or both gram negative),then the species of the co-infection can be identified. If aco-infection is present and is of a different category (infection is GPand co-infection is GN or fungal for example), the genus of the fungalco-infection can be identified and the category (Gram-negative) of theco-infection can be identified. If the co-infection is of a differentcategory than the infection, the species of the co-infection can beidentified by applying a two-step detection method. Further, the methodsand systems identify the genus of an organism which is likely to be acontaminating organisms from a blood draw. Further, the methods andsystems identify the presence of Propionibacterium acnes, Staphylococcusepidermidis, Micrococcus, Lactobacillus or Corynebacterium which arede-escalation targets.

This technical solution solves the problem stated above, namely, itenables the detection and/or identification of a human pathogen (i.e.,the amplified human pathogen hybridized to a signal and capture probe isdetected) with elimination and/or reduction of false positives due tocontamination, thereby enabling informed decisions to be made regardingantibiotic stewardship.

In an embodiment, the application of PCR using a multiplex PCR methodenables a substantial reduction in electrochemical detection ofcontaminating pathogen and/or genetic material present in the samplewhile allowing infectious bacteria and/or fungi to be detected.Detection occurs when the infectious bacteria and/or fungi are amplifiedand the amplicon hybridizes with a signal and/or capture probe.

One aspect of the invention discloses methods and devices foridentifying which of a plurality of target nucleic acids is in a sample.The disclosed methods comprise providing a sample to the cartridge,providing PCR regents (including, but not limited to primers, dNTPs, DNApolymerase, exonucleases, etc.) for amplifying a locus from a differentone of a plurality of target nucleic acid sequence to the sample,subjecting the sample to amplification conditions through a number ofamplification cycles, detecting whether amplification has occurred, andidentifying the target nucleic acid present in the sample whereinidentifying comprises determining if the target nucleic acid ishybridized to signal and capture probes. In one embodiment, non-uniformPCR cycling is used in a single cartridge, i.e., a single cartridge maycycle a sample and a first set of primers 30 times and cycle the sampleand a second different set of primers 35 times (based on using differentlocations on the cartridge; reference is made to FIG. 15).

The overall method of the invention is preferably substantially specificregarding the identification of the pathogen. An infectious pathogen canbe identified by its species. A co-infectious pathogen which is of thesame type as the infection (both are gram-positive, both aregram-negative or both are fungal) can be identified by its species. Aco-infectious pathogen which is not the same type as the infection (theinfection is gram-positive and co-infection is gram-negative or fungal;the infection is gram-negative and the co-infection is gram-positive orfungal), can be identified by its species.

Co-infectious pathogens not being a member of a predetermined group(pan-fungal or pan-gram-negative for the BCID-GP panel; or pan-fungal orpan gram-positive for the BCID-GN panel) are not identified because thesteps performed with the reagents are adjusted to not detect pathogensnot belonging to that group. In a preferred embodiment, 20-30 infectiouspathogens can be identified on a single cartridge by its species orgenus using a single PCR run while simultaneously being able todistinguish between systemic infection and punitive contamination. In apreferred embodiment, 30-40 or 40-50 infectious pathogens can beidentified on a single cartridge by its species or genus using a singlePCR run while simultaneously being able to distinguish between systemicinfection and punitive contamination. In a preferred embodiment, atleast 20, 20-60; 30-40 or 40-50 infectious pathogens can be identifiedon a single cartridge by its species or genus using a single PCR runwhile simultaneously being able to distinguish between systemicinfection and punitive contamination and while simultaneouslyidentifying fungal and bacteria co-infections by genus (fungal) orcategory (gram positive or gram negative).

Purification

Purification, partial purification or isolation of nucleic acids (e.g.DNA) from the clinical sample after gram staining is not needed toachieve sufficient sensitivity for detecting an infection while notdetecting contaminants in the sample. Particularly, the nucleic acidsneed not be separated from proteins, sugars, and salts present in theoriginal clinical sample. It is not necessary to partially or evencompletely isolate nucleic acid from the clinical sample after gramstaining.

Alternatively, the nucleic acid target (genome, gene or gene fragment(e.g., a restriction fragment) of the pathogen) may be in a purified, orin an isolated form.

Alternatively, the sample may be treated with a compound whichhydrolyzes nucleic acids aka a nuclease before amplification.Specifically, the sample may be treated with DNase I, Benzonase, or S1nuclease before amplification, preferably before cell lysis.

Primer Amplification

In general, the design of amplification primers is performed on thebasis of available sequence information with regard to the pre-selectedtarget nucleic acid sequence regions of the specific pathogenicgram-positive bacteria to be amplified as well as with regard to thehomologous sequences of those gram-positive and gram-negative bacteria,which shall not be amplified. More precisely, the set or sets ofamplification primers are selected in such a way that there is a maximumsequence complementarity with respect to all target nucleic acidsequences of the selected predetermined pathogenic gram-positivebacteria species or genus, and, on the other hand, a minimum sequencecomplementarity with respect to nucleic acid sequences of all othernon-selected gram-positive bacteria, gram-negative bacteria, i.e. thosenot belonging to the predetermined group or not being pathogenic, aswell as fungi. The same method is applied to the BCID-GN cartridge andBCID-FN cartridge.

The invention surprisingly shows that the analysis of fungi is possiblein a single PCR reaction, without a nested PCR approach, in such amanner that a highly sensitive and very specific method is provided.This is surprising as generally, due to the slower growth of fungalinfections, the fungal pathogens are present in lower amounts in thesample, and, thus, signal from contaminants can compete with the actualsignal from the fungal pathogen. Previous attempts at PCR followed bydetection have been bothered by high levels of false positives caused bycontaminating pathogen and/or genetic material present in the sampleand/or media bottle. See U.S. Application no. 2015/0232916. Theinvention is, therefore, the first described single-run multiplex PCRmethod for discrimination between contaminating pathogen and/or geneticmaterial present in the sample and infectious pathogen combined withdiscrimination between gram-positive pathogens, gram-negative bacterialpathogens, and fungal pathogens in said sample as well as antimicrobialresistance genes. The complexity of the present method is significantlyreduced compared to alternative amplification schemes describedpreviously, thereby increasing the user friendliness and reproducibilitycompared to those methods of the prior art.

In an embodiment of the invention, the method is characterized in thatthe PCR reaction comprises oligonucleotides that bind a DNA/nucleic acidsequence of a bacterial pathogen. In another embodiment, the method ofthe invention is characterized in that the oligonucleotides capable ofbinding a sequence of a bacterial pathogen enable discrimination betweengram-positive and gram-negative bacteria. In one embodiment the methodof the invention is characterized in that the oligonucleotides capableof binding a DNA/nucleic acid sequence of a bacterial pathogen which,once amplified, attach to probes labeled so as to be distinguished fromeach other.

In an embodiment, the oligonucleotides designed for DNA amplificationare able to amplify genetic material from a single pathogenic orpotentially pathogenic bacteria (i.e. specific for sequence variation ofa particular species or genus of a gram-positive bacteria) allowingdetection of a specific species or genus of gram-positive bacterialinfection and are run with oligonucleotides that detect the fungal genusor gram-negative genus and, as a result, is a broad-band, gram-negativebacterial and fungal detection method. Likewise, in an embodiment, theoligonucleotides designed for DNA amplification allow detection of aspecific gram-negative bacterial infection (i.e. specific for sequencevariation of a particular species or genus of a gram-negative bacteria)and are run with oligonucleotides that detect the fungal genus or thegram-positive bacteria genus or species but do not identifygram-positive or fungal infections by genus or species and, as a result,is a broad-band, gram-positive bacterial or fungal and detection method.

In an embodiment of the invention, the method is characterized in thatthe PCR reaction comprises oligonucleotides that bind a DNA sequence ofa fungal pathogen. In one embodiment the method of the invention ischaracterized in that the oligonucleotides capable of binding a DNAsequence of a fungal pathogen which, once amplified, attach to probeslabeled so as to be distinguished from each other.

In a surprising manner, the oligonucleotides designed for fungal DNAamplification are able to amplify genetic material from a singlepathogenic or potentially pathogenic fungi (i.e. specific for sequencevariation of a particular species or genus of fungi) allowing detectionof a specific fungal infection and do not detect contaminating pathogenand/or genetic material present in the sample.

Probes

In one embodiment the method of the invention is characterized in thatthe signal probes comprise electrochemical labels, wherein multipleprobes may be identified and differentiated from one another on thebasis of distinct labels that emit electrical signals at differentvoltages from each other; see for example, U.S. Pat. No. 7,935,481 andU.S. patent application Ser. No. 10/137,710 (which are herebyincorporated by reference in their entirety) which disclose a pluralityof probes each with at least one ETM with a unique redox potential. Thisis analogous to the “two color” or “four color” idea of competitivehybridization, and is also analogous to sequencing by hybridization.Probes and labels may be selected as required depending on the deviceused for analysis and the sample to be assessed as known by thoseskilled in the art. Preferred labels for signal probes include ferroceneand ferrocene derivatives. Ferrocene undergoes many reactionscharacteristic of aromatic compounds, enabling the preparation ofsubstituted derivatives. Ferrocene derivatives (such as N6, QW56, andQW80) and are generally covalently attached to the signal probes.

Single PCR Run

In an embodiment the method of the invention is carried out in a singlemultiplex, end point (PCR) reaction, otherwise known as a single PCR run(to be distinguished from nested PCR).

The invention is therefore characterized by the reduced number of PCRruns (single run) employed in the method compared to the prior art. Theinvention is therefore characterized by the reduced number of primersemployed in the method compared to the prior art. The invention istherefore characterized by the reduced number of PCR runs (single run),PCR cycles (40, 35 or 30) and primers employed in the method compared tothe prior art.

The invention is characterized in that some targets are detected with a35 PCR cycle while other targets are detected with reduced PCR cycling(30) but there is a single PCR run. As such, the invention ischaracterized in that there is a single PCR run of the sample in asingle cartridge.

Detuning

In recent years, there has been a growing demand for quick and highlysensitive systems for detecting infectious diseases. New systems use areverse transcription-PCR and nested PCT to increase assay sensitivity.But with increased sensitivity, false positive results may occur.Indeed, there is a risk of false positives for Pseudomonas aeruginosaand Enterococcus results using bioMérieux BacT/ALERT SN StandardAnaerobic Blood Culture Bottles (Catalog Number 259790). See FIG. 23.

The art teaches that “tuning” the number of PCR cycles when using nestedPCT can minimize false positive calls from background contamination,cross-reactivity (which can be problematic in a highly multiplexedreaction), and other extraneous amplification. See U.S. Patentapplication no. US20150232916 which is herein incorporated by referencein its entirety. However such approaches require nested PCR to achievethe necessary sensitivity. Indeed, a single run PCR with reduced cycling(less than 40 cycles) may be insufficient to detect some organismsbecause they amplify much later, because of slower growth in culture,less efficient PCR, or because there are fewer copies of the targetsequence in a positive blood culture. Indeed, the BCID-FP panel cycles40 times because fungi is known to grow slower in culture and the assayis detuned by having primer mismatches or having dual zone detection.Additionally, a single run PCR with reduced cycling (less than 40cycles) could result in false negatives because a single PCR run isinsufficient to amplify and detect the organism. It was surprising andunexpected that the balance of sensitivity (detection of low titerinfectious organisms) and non-detection of contaminates could beachieved in a single PCR run using end-point PCR not nested PCR.

Prior to Applicant's discovery, the vast number of organisms' nucleicacid in blood culture bottles was not recognized in the field. Table 5below shows that over 20 contaminating organisms' DNA is found in bloodculture bottles. It was further surprising that the assays could bedetuned in such a way that only clinically relevant detection wasachieved given the vast number of organisms' DNA detected in bloodculture bottles. It was further surprising that the assays could bedetuned in such a way that only clinically relevant detection wasachieved regardless of the blood culture bottle used (sensitivity is notlimited to a particular blood culture bottle type).

Indeed, the ability to de-tune the assay is hampered by the system'sfour-track PCR configuration. With such a system only two PCR cycles canbe run at a time because when lanes 1-4 are being denatured, lanes 5-8are being amplified (See FIG. 15). It was surprising that clinicallyrelevant infection could be distinguished from background contaminationfor the vast number of contaminating organisms detected in empty bottlesusing only 2 PCR cycling conditions (a.k.a., a single PCR run with twomismatched PCR cycles) in the cartridge.

The invention is characterized in that the electrochemical detectionsystem employed, needed to be made less sensitive, “detuned,” toeliminate or reduce detection of contaminants while remaining sensitiveenough to detect clinically relevant infection. Detuning was achieved byreducing the PCR cycles in each single PCR run, increasing or decreasingthe primer concentration, thresholding, primer mismatch and/or requiringone pathogen be detected in two detection zones. While the molecularbiology techniques used to detune were known, no one had applied them inthe context of electrochemical detection in a single run PCR to detectpathogens but not background contamination. In this way, applicants wereable to reduce false positives from bottle contaminates to less than 5%,preferably less than 4%, preferably less than 3%, preferably less than2%, preferably less than 1%, preferably less than 0.5%, preferably lessthan 0.1%, preferably less than 0.0.5%, preferably between 0.05%-5%,preferably between 0.5%-1%, preferably between 0.5%-3%, preferablybetween 0.01%-1%. Further, prior to Applicant, no one had applied asingle run end-point PCR utilizing two mismatched PCR cycles to detectclinically relevant pathogens but not background contamination. Prior toApplicant, no one had applied a single run end-point PCR utilizing twomismatched PCR cycles to detect about 23 clinically relevant pathogensby genus and identify about 15 by their species but not backgroundcontamination. Prior to Applicant, no one had applied a single runend-point PCR utilizing two mismatched PCR cycles to detect 15-30clinically relevant pathogens by genus and identify about 10-30 by theirspecies but not background contamination. Another problem associatedwith nested PCR is that because there are two amplifications the numberof reagents and primers needed is high compared to a single PCR run. Assuch, nested PCT systems cannot detect as many organisms as a systemutilizing a single PCR run. As such, nested PCT systems tend to befocused on genus calls as opposed to species calls, like the invention.As such, only a broadband antibiotic therapeutic approach is possiblewhen a nested PCT system identifying only genus calls is used, whichmay, in fact, be poorly suited for the particular pathogen.

The invention can be further understood by the following numberedparagraphs:

Paragraph 1. A method for identifying which of a plurality of organismsis in a sample, comprising: (a) providing a plurality of sample wells,each sample well provided with a portion of the sample and primers foramplifying a target nucleic acid sequence from a different one of theplurality of organisms, subjecting the plurality of sample wells to asingle amplification having a number of predetermined amplificationcycles, detecting whether amplification has occurred in each of a secondset of the plurality of sample wells, identifying at least one organismpresent in the sample.

Paragraph 2: The method of paragraph 1, further comprising subjectingthe plurality of sample wells to a single amplification conditionwherein the number of predetermined amplification cycles can bemismatched.

Paragraph 3: The method of paragraph 1, further comprising subjectingthe plurality of sample wells to a single amplification conditionwherein the number of predetermined amplification cycles is 30 or 35.

Paragraph 4. A method for identifying which of a plurality of organismsis in a sample, comprising: (a) providing a plurality of sample wells,each sample well provided with a portion of the sample and primers foramplifying a target nucleic acid sequence from a different one of theplurality of organisms, subjecting the plurality of sample wells to asingle amplification having a heterogeneous number of amplificationcycles, detecting whether amplification has occurred in each of a secondset of the plurality of sample wells, identifying at least one organismpresent in the sample.

Antibiotic Stewardship

In an embodiment, the method of the invention is characterized in thatthe sample is obtained from a subject exhibiting one or more symptoms ofsystemic inflammatory response syndrome (SIRS), sepsis, severe sepsisand/or septic shock. A significant benefit of this approach is theability to subsequently prescribe an appropriate medicament duringtreatment. In light of the knowledge regarding bacterial (gram-positive,gram-negative) or fungal pathogen presence (as well as its resistanceprofile), an appropriate antibiotic or an appropriate anti-fungal can beselected for treatment, thereby avoiding potentially useless antibiotictreatments and associated financial, health and environmentaldisadvantages.

Patient care and antibiotic stewardship would be advanced by developmentand application of rapid diagnostics that provide accurate and timelyinformation as to the nature of the infecting pathogen, includingwhether it is gram-positive bacterial, gram-negative bacterial, fungaland its resistance profile.

The BCID-GP, GN and FP Panels also includes several targets fororganisms known to be common blood culture contaminants to aid inrapidly ruling out blood culture contamination. Other molecular panelsinclude only coagulase negative Staphylococcus (CoNS) while the BCIDpanel includes Bacillus subtilis group, Corynebacterium, Lactobacillusgroup, Micrococcus and Propionibacterium acnes in addition to CoNS.

This is important because studies have shown that up to 15-30% ofpositive blood cultures may be contaminants depending on the lab. Sobeing able to quickly determine a contaminant from a true infectionmeans clinicians can more rapidly de-escalate unnecessary antibioticsand get patients out of the hospital quickly, instead of waiting 2-3days for identification and antimicrobial susceptibility testing (AST).That also limits the adverse outcomes from unnecessary antibiotics.

Propionibacterium acnes, Staphylococcus epidermidis, Micrococcus,Lactobacillus or Corynebacterium can be true pathogens but in order forclinicians to determine a true pathogen from a contaminant they willlook at several factors including: whether or not the patient isimmunocompromised, the number of blood culture bottles that rangpositive (if more than one, it is considered a true pathogen), time tobottle positivity compared to other bottles (If a bottle rings positivelater than others it is often considered a contaminant because thebacterial load is generally lower) and other clinical symptoms.

Specifically, when contamination occurs at blood draw there is apositive gram stain and the physician can begin treatment.Propionibacterium acnes, Staphylococcus epidermidis, Micrococcus,Lactobacillus and Corynebacterium on the BCID-GP GN and FP panels arereferred to as “de-escalation targets.” When these targets are positiveon the BCID panels and/or gram stain, the physician can evaluate whetherthe organism detected is likely the result of a blood infection orsample contamination. Sample contamination is especially likley whenorganisms such as Propionibacterium acnes, Staphylococcus epidermidis,Micrococcus, Lactobacillus and Corynebacterium are identified in onepatient sample but not the other. When these are identified thephysician or laboratory can verify infection by a second method.Although, sometimes the detection of these targets are technically acontamination, the identification of these on the BCID-GP GN and FPpanels leads to clinically actionable data because the physician canevaluate whether the organism detected is likely the result of a bloodinfection or sample contamination.

In one embodiment the method of the invention is characterized in thatpatient treatment is altered or started based on the results from aBCID-GP, BCID-GN, or BCID-FP assay.

In one embodiment, an initial analysis is performed on a first sample todetermine whether or not one or more common contaminants (such asPropionibacterium acnes, Staphylococcus epidermidis, Micrococcus,Lactobacillus or Corynebacterium) are present in a sample. If thisinitial analysis indicates that the common contaminant is present, asecond analysis is performed to determine if the common contaminant ispresent in a second sample. If the common contaminant is not present ina second analysis then it is presumed the first sample was contaminated.

The invention can be further understood by the below numberedparagraphs:

Paragraph 1: An in vitro method for the detection and/or identificationof a hybridization complex comprising a human pathogen and/or geneticmaterial thereof hybridized to a signal probe and a capture probecomprising: subjecting a sample comprising or suspected of comprising ahuman pathogen and/or genetic material thereof to a single multiplexpolymerase chain reaction (PCR), wherein said method comprisesamplification of PCR products under conditions appropriate for thesubstantial reduction in detection of contaminating pathogen and/orgenetic material present in the sample from blood culture bottles, anddetecting the binding between the human pathogen and/or genetic materialthereof and the signal probe and a capture probe and detecting thebinding between Propionibacterium acnes, Staphylococcus epidermidis,Micrococcus, Lactobacillus or Corynebacterium and the signal probe and acapture probe.

Paragraph 2: The in vitro method of paragraph 1, wherein the detectionof Propionibacterium acnes, Staphylococcus epidermidis, Micrococcus,Lactobacillus or Corynebacterium is compared to a second detectionmethod and/or factor such as whether or not the patient isimmunocompromised, the number of blood culture bottles that rangpositive (if more than one, it is considered a true pathogen), time tobottle positivity compared to other bottles (If a bottle rings positivelater than others it is often considered a contaminant because thebacterial load is generally lower) and other clinical symptoms.

Paragraph 3: The in vitro method of paragraph 2, wherein if the seconddetection method does not detect Propionibacterium acnes, Staphylococcusepidermidis, Micrococcus, Lactobacillus or Corynebacterium then thedetection of Propionibacterium acnes, Staphylococcus epidermidis,Micrococcus, Lactobacillus or Corynebacterium rules out infection byPropionibacterium acnes, Staphylococcus epidermidis, Micrococcus,Lactobacillus or Corynebacterium.

Paragraph 4: An in vitro method for ruling out infection in a patientcomprising: subjecting a sample comprising or suspected of comprising ahuman pathogen and/or genetic material thereof and a contaminate to asingle multiplex polymerase chain reaction (PCR), and detecting thebinding between the human pathogen and/or genetic material thereof andthe signal probe and a capture probe and detecting the binding betweenPropionibacterium acnes, Staphylococcus epidermidis, Micrococcus,Lactobacillus or Corynebacterium and a signal probe and a capture probewherein the detection of Propionibacterium acnes, Staphylococcusepidermidis, Micrococcus, Lactobacillus or Corynebacterium rules outinfection by Propionibacterium acnes, Staphylococcus epidermidis,Micrococcus, Lactobacillus or Corynebacterium.

Paragraph 5: An in vitro method for determining if a sample iscontaminated comprising: subjecting a sample comprising or suspected ofcomprising a human pathogen and/or genetic material thereof to a singlemultiplex polymerase chain reaction (PCR), and detecting the bindingbetween the human pathogen and/or genetic material thereof and thesignal probe and a capture probe and detecting the binding betweenPropionibacterium acnes, Staphylococcus epidermidis, Micrococcus,Lactobacillus or Corynebacterium and a signal probe and a capture probewherein the detection of Propionibacterium acnes, Staphylococcusepidermidis, Micrococcus, Lactobacillus or Corynebacterium indicates thesample was likely contaminated. In embodiments, the method can furtherdifferentiate between gram-positive bacteria, gram-negative bacteria,fungi and determinates for antimicrobial resistance. In embodiments, themethod can further identify the gram-positive bacteria species,gram-negative bacteria species, or fungi species. In embodiments, themethod can further identify a co-infection if present in the sample bygenus or type (gram-positive bacteria, gram-negative bacteria).

Paragraph 6: A method for testing a blood sample for the presence of apossible contaminant and a human pathogen and/or genetic materialthereof, that reduces the risk of a false positive indication forcontaminations frequently present in blood samples, the methodcomprising the steps of: obtaining a sample; subjecting the samplecomprising or suspected of comprising a contamination or human pathogenand/or genetic material thereof to a single multiplex polymerase chainreaction (PCR), and detecting the binding between the possiblecontaminant and the signal probe and a capture probe; and detectingbinding between the human pathogen and/or genetic material thereof andthe signal probe and a capture probe wherein when binding betweenPropionibacterium acnes, Staphylococcus epidermidis, Micrococcus,Lactobacillus or Corynebacterium and a signal probe and a capture probeis detected, there is a possible contamination. In embodiments, themethod can further differentiate between gram-positive bacteria,gram-negative bacteria, fungi and determinates for antimicrobialresistance. In embodiments, the method can further identify thegram-positive bacteria species, gram-negative bacteria species, or fungispecies. In embodiments, the method can further identify a co-infectionif present in the sample by genus or type (gram-positive bacteria,gram-negative bacteria).

Paragraph 7: A method for testing a blood sample for the presence of apossible contaminant and a human pathogen and/or genetic materialthereof, that reduces the risk of a false positive indication forcontaminations frequently present in blood samples, the methodcomprising the steps of: obtaining a sample; subjecting the samplecomprising or suspected of comprising a contamination or human pathogenand/or genetic material thereof to a single multiplex polymerase chainreaction (PCR) wherein said method comprises amplification of PCRproducts under conditions appropriate for the substantial reduction indetection of contaminating pathogen and/or genetic material present inthe sample as a result of blood culture bottle contamination, anddetecting the binding between the possible contamination and the signalprobe and a capture probe; and detecting binding between the humanpathogen and/or genetic material thereof and the signal probe and acapture probe wherein when binding between the possible contamination(Propionibacterium acnes, Staphylococcus epidermidis, Micrococcus,Lactobacillus or Corynebacterium) and a signal probe and a capture probeis detected, there is a possible contamination.

Paragraph 8: A method for identifying the presence of a possiblecontaminant in a sample comprising: obtaining a sample; subjecting thesample comprising or suspected of comprising a contamination and/orhuman pathogen and/or genetic material thereof to a single multiplexpolymerase chain reaction (PCR) wherein said method comprisesamplification of PCR products under conditions appropriate for thesubstantial reduction in detection of the contaminating pathogen and/orgenetic material present in the sample as a result of blood culturebottle contamination, and detecting the binding between the possiblecontaminant and the signal probe and a capture probe; and detectingbinding between the human pathogen and/or genetic material thereof andthe signal probe and a capture probe wherein when binding between thepossible contaminant (Propionibacterium acnes, Staphylococcusepidermidis, Micrococcus, Lactobacillus or Corynebacterium) and a signalprobe and a capture probe is detected, indicates the presence of apossible contaminant is identified. In embodiments, the method canfurther differentiate between gram-positive bacteria, gram-negativebacteria, fungi and determinates for antimicrobial resistance. Inembodiments, the method can further identify the gram-positive bacteriaspecies, gram-negative bacteria species, or fungi species. Inembodiments, the method can further identify a co-infection if presentin the sample by genus or type (gram-positive bacteria, gram-negativebacteria).

Paragraph 9: A method for identifying the presence of a de-escalationtarget in a sample comprising: obtaining a sample; subjecting the samplecomprising or suspected of comprising a human pathogen and/or geneticmaterial thereof to a single multiplex polymerase chain reaction (PCR)wherein said method comprises amplification of PCR products underconditions appropriate for the substantial reduction in detection of thecontaminating pathogen and/or genetic material present in the sample asa result of blood culture bottle contamination, and detecting thebinding between the de-escalation target and the signal probe and acapture probe; and detecting binding between the human pathogen and/orgenetic material thereof and the signal probe and a capture probe. Inembodiments, the method can further differentiate between gram-positivebacteria, gram-negative bacteria, fungi and determinates forantimicrobial resistance. In embodiments, the method can furtheridentify the gram-positive bacteria species, gram-negative bacteriaspecies, or fungi species. In embodiments, the method can furtheridentify a co-infection if present in the sample by genus or type(gram-positive bacteria, gram-negative bacteria).

Paragraph 10: A microfluidic device for the detection and/oridentification of a human pathogen and/or genetic material thereofcomprising: a mixture of oligonucleotides and reagents for carrying outa single nucleic acid amplification reaction capable of distinguishingbetween clinically relevant amplification and amplification from bloodculture bottle contamination and further capable of identifying thepresence of a de-escalation target in a sample wherein the de-escalationtarget is Propionibacterium acnes, Staphylococcus epidermidis,Micrococcus, Lactobacillus or Corynebacterium. In embodiments, thedevice can further differentiate between gram-positive bacteria,gram-negative bacteria, fungi and determinates for antimicrobialresistance. In embodiments, the device can further identify thegram-positive bacteria species, gram-negative bacteria species, or fungispecies. In embodiments, the device can further identify a co-infectionif present in the sample by genus or type (gram-positive bacteria,gram-negative bacteria).

Paragraph 11: A detection report identifying the presence of a possiblecontaminant in a sample wherein the possible contaminant isPropionibacterium acnes, Staphylococcus epidermidis, Micrococcus,Lactobacillus or Corynebacterium.

Paragraph 12: A detection report identifying the presence of a possiblecontaminant in a sample wherein the possible contaminant isPropionibacterium acnes, Staphylococcus epidermidis, Micrococcus,Lactobacillus or Corynebacterium and further identifying of a humanpathogen and/or genetic material thereof comprising gram-positivebacteria, gram-negative bacteria or fungi. In some embodiments thegram-positive bacteria or gram-negative bacteria or fungi is identifiedby its species and a co-infection if present is identified by its genusor type (gram-positive or negative).

Paragraph 13: A detection report identifying the presence of ade-escalation target wherein the de-escalation target isPropionibacterium acnes, Staphylococcus epidermidis, Micrococcus,Lactobacillus or Corynebacterium and further identifying of a humanpathogen and/or genetic material thereof comprising gram-positivebacteria, gram-negative bacteria or fungi. In some embodiments thegram-positive bacteria or gram-negative bacteria or fungi is identifiedby its species and a co-infection if present is identified by its genusor type (gram-positive or negative).

Paragraph 14: A method for distinguishing between unwanted contamination(from blood culture bottles) and possible contaminant (from blood draw)but which may be a clinically relevant infection the method comprisingobtaining a sample; subjecting a sample comprising or suspected ofcomprising a human pathogen and/or genetic material thereof to a singlemultiplex polymerase chain reaction (PCR), wherein said method comprisesamplification of PCR products under conditions appropriate for thesubstantial reduction in detection of unwanted contaminating pathogenand/or genetic material present in the sample and appropriate for thedetection of possible contaminant in the sample wherein the possiblecontaminant is Propionibacterium acnes, Staphylococcus epidermidis,Micrococcus, Lactobacillus or Corynebacterium. In embodiments, themethod can further differentiate between gram-positive bacteria,gram-negative bacteria, fungi and determinates for antimicrobialresistance. In embodiments, the device can further identify thegram-positive bacteria species, gram-negative bacteria species, or fungispecies. In embodiments, the device can further identify a co-infectionif present in the sample by genus or type (gram-positive bacteria,gram-negative bacteria).

Paragraph 15: A device for distinguishing between unwanted contamination(from blood culture bottles) and possible contaminant (from blood draw)but which may be a clinically relevant infection in a sample the devicecomprising: a mixture of oligonucleotides and reagents for carrying outa single nucleic acid amplification reaction capable of distinguishingbetween a possible contaminant and unwanted contamination. The devicecan further include a mixture of oligonucleotides and reagents forcarrying out a single nucleic acid amplification reaction capable ofdistinguishing between clinically relevant pathogen and unwantedcontamination and possible contamination. In embodiments, the device canfurther differentiate between gram-positive bacteria, gram-negativebacteria, fungi and determinates for antimicrobial resistance. Inembodiments, the device can further identify the gram-positive bacteriaspecies, gram-negative bacteria species, or fungi species. Inembodiments, the device can further identify a co-infection if presentin the sample by genus or type (gram-positive bacteria, gram-negativebacteria).

Method(s) of the Invention(s)

In one embodiment the method of the invention comprises or consists ofthe following steps: a) providing a sample, preferably a blood culture,blood, serum or plasma sample, b) after nucleic acid extraction,bringing said sample into contact with a mixture of oligonucleotides andreagents (as well as phenol red) for carrying out a nucleic acidamplification reaction, c) carrying out a single nucleic acidamplification reaction, and d) detecting and evaluating theamplification products generated as a result of said single nucleic acidamplification reaction.

In general, the method is suitable for detection of a bacteria or fungifrom a sample. In general, the method is suitable for identification ofa gram-positive bacteria, gram-negative bacteria or fungi from a sample.The identification of a pathogen may occur such that the detectionreport provides “fungal”, “gram-positive” or “gram-negative” as anappropriate result. The identification of a pathogen may occur such thatthe detection report provides the fungal species name, gram-positivebacteria species name or gram-negative bacteria species name as anappropriate result. The identification of a pathogen may occur such thatthe detection report provides the fungal species or genus name,gram-positive bacteria species or genus name or gram-negative bacteriaspecies or genus name as an appropriate result.

The identification of a pathogen may occur such that the detectionreport provides the gram-positive bacteria species or genus name, thefungal genus name and/or identifies gram-negative bacteria detection asan appropriate result. The identification of a pathogen may occur suchthat the detection report provides the gram-negative bacteria species orgenus name, the fungal genus name and/or identifies gram-positivebacteria detection as an appropriate result.

Below summarizes the types of calls/reports for each BCID panel.

A method for reducing or eliminating false positives comprising thesteps of: a) providing a sample b) after nucleic acid extractioncontacting the sample with a mixture of oligonucleotides and reagentsfor carrying out a single nucleic acid amplification reaction capable ofdistinguishing between clinically relevant pathogen and endogenous orcontaminating DNA thereby reducing or eliminating false positives. Insome embodiments, after using the methods of the invention, falsepositives range from 0.001-5%, 0.001-3%, 0.001-1%, 0.05-1%, 0.1-1%.

A method for detecting the presence of a pathogen of interest in asample, comprising the steps of: a) providing sample comprising apathogen; b) after nucleic acid extraction contacting the sample with amixture of oligonucleotides and reagents (including phenol red) forcarrying out a single nucleic acid amplification reaction c) amplifyunder conditions appropriate for pathogen replication and thesubstantial reduction of endogenous or contaminating DNA replication;and d) detecting the presence of amplified pathogen in the sample.

The methods of detection may be carried out by amplification of thegenetic material, by hybridization of the genetic material witholigonucleotides or by a combination of amplification and hybridization.A significant advantage of the invention is that the amplification stepmay be performed under similar or uniform amplification conditions foreach pathogen species or genus. As such, amplification of each pathogenspecies or genus may be performed simultaneously. Detection of thegenetic material may also advantageously be performed under uniformconditions.

It is an object of the invention to provide a method of detecting anucleic acid sequence which reduces the number of false positivesresulting from nucleic acid contamination in the sample (i.e., organismsor nucleic acid found in the blood culture bottle media). The presentmethod increases the accuracy of the procedure without sacrificingclinically relevant sensitivity.

It is another object of the invention to provide a method of detecting anucleic acid sequence which obviates the necessity to select a signalingthreshold.

It is another object of the invention to provide methods and systems todetect nucleic acid sequences which identify blood culture drawcontamination.

Device(s) of the Invention(s)

A microfluidic device for detecting a genetic material, comprising: amixture of oligonucleotides and reagents (including phenol red) forcarrying out a single nucleic acid amplification reaction capable ofdistinguishing between clinically relevant amplification andamplification from other sources such as from contamination. Wherein themixture of oligonucleotides and reagents for carrying out a singlenucleic acid amplification reaction is further capable of distinguishingbetween gram-positive, gram-negative, or fungal infection. Wherein themixture of oligonucleotides and reagents for carrying out a singlenucleic acid amplification reaction is further capable of identifyingantimicrobial resistance. Wherein the mixture of oligonucleotides andreagents for carrying out a single nucleic acid amplification reactionis further capable of identifying the species of the infection andspices of a co-infection. Wherein the mixture of oligonucleotides andreagents for carrying out a single nucleic acid amplification reactionis further capable of identifying the species or genus of the infectionand spices or genus of a co-infection. Wherein the mixture ofoligonucleotides and reagents for carrying out a single nucleic acidamplification reaction is further capable of identifying the species orgenus of the infection and spices or type (gram-positive or gramnegative) of co-infection.

A cartridge comprising: a mixture of oligonucleotides and reagents forcarrying out a single nucleic acid amplification reaction capable ofdistinguishing between clinically relevant amplification andamplification from other sources such as from background contamination.Wherein the mixture of oligonucleotides and reagents for carrying out asingle nucleic acid amplification reaction is further capable ofdistinguishing between gram-positive, gram-negative, or fungalinfection. Wherein the mixture of oligonucleotides and reagents forcarrying out a single nucleic acid amplification reaction is furthercapable of identifying antimicrobial resistance. Wherein the mixture ofoligonucleotides and reagents for carrying out a single nucleic acidamplification reaction is further capable of identifying the species ofthe infection and genus of a co-infection. Wherein the mixture ofoligonucleotides and reagents for carrying out a single nucleic acidamplification reaction is further capable of identifying the species orgenus of the infection and spices or genus of a co-infection. Whereinthe mixture of oligonucleotides and reagents for carrying out a singlenucleic acid amplification reaction is further capable of identifyingthe species or genus of the infection and spices or type (gram-positiveor gram negative) of co-infection.

In some embodiments, gram-positive and gram-negative primers are in thesame multiplex primer pool. In some embodiments, gram-positive andfungal primers are in the same multiplex primer pool. In someembodiments, gram-negative and fungal primers are in the same multiplexprimer pool.

Gram-Positive

The Gram-Positive (BCID-GP) Panel is a fully automated, qualitative,nucleic acid, multiplex in vitro diagnostic test for the simultaneousqualitative detection and identification of multiple potentiallypathogenic gram-positive bacterial organisms and select determinants ofantimicrobial resistance in positive blood culture. In addition, theBCID-GP Panel also detects but does not differentiate Gram-Negativebacteria (Pan Gram-Negative assay giving a gram-negative call) andseveral Candida species (Pan Candida assay giving a Candida call)present in co-infections. The BCID-GP Panel is performed directly onblood culture samples identified as positive by a continuouslymonitoring blood culture system that demonstrate the presence oforganisms as determined by Gram stain.

The BCID-GP Panel contains assays for the detection of geneticdeterminants of resistance to methicillin (mecA and mecC) and vancomycin(vanA and vanB) to aid in the identification of potentiallyantimicrobial resistant organisms in positive blood culture samples. Theantimicrobial resistance gene detected may or may not be associated withthe agent responsible for the disease.

The BCID-GP Panel also contains targets designed to detect a broad rangeof organisms with a potentially misleading Gram stain result ororganisms that may be missed by Gram staining altogether for example inthe case of co-infections. These include a broad Pan Gram-Negative assayas well as a Pan Candida assay, both of which may provide data tofacilitate the correct testing algorithm. As such, the presentdisclosure relates to methods and systems for a) distinguishing betweencontamination and gram-positive bacterial infection, b) distinguishingbetween gram-positive bacterial species infection; c) distinguishingbetween some gram-positive bacterial species and some gram-positivegenus infection(s); d) identifying but not differentiating gram-negativebacterial infection and fungal infection. The present disclosure furtherrelates to methods and systems for identifying a pathogen that is likelya contamination from the blood draw.

The following bacterial organisms and resistance marker genes areidentified using the BCID-GP Panel: Bacillus cereus group,Staphylococcus epidermidis, Bacillus subtilis group, Staphylococcuslugdunensis, Corynebacterium spp., Streptococcus, Enterococcus,Streptococcus agalactiae, Enterococcus faecalis, Streptococcus anginosusgroup, Enterococcus faecium, Streptococcus pneumonia, Lactobacillus,Streptococcus pyogenes, Listeria, Pan Gram-negative target (at leastEnterobacteriaceae, Acinetobacter, Pseudomonas, Bacteroides,Stenotrophomonas), Listeria monocytogenes, Pan Candida target (Candidaalbicans, Candida glabrata, Candida krusei, Candida parapsilosis),Micrococcus, Propionibacterium acnes, Staphylococcus, Staphylococcusaureus, mecA, mecC, vanA, and vanB. Table 1 below shows that reportedtarget call and the target species detected. Stated another way, somespecies are detected by the BCID-GP panel (“Targets detected” in Table 1below) but not identified by the species in the call (report); instead,the call/report identifies the genus. Some species are detected by theBCID-GP panel (“Targets detected” in Table 1 below) and are identifiedby the species in the call (report). Some organisms can generate boththe genus and species call.

TABLE 1 Analytes Detected by the BCID-GP Panel Calls Reported TargetTargets Detected 1 Streptococcus agalactiae Streptococcus agalactiae 2Streptococcus anginosus Streptococcus constellatus group Streptococcusintermedius Streptococcus anginosus 3 Streptococcus pneumoniaeStreptococcus pneumoniae 4 Streptococcus pyogenes Streptococcus pyogenes5 Staphylococcus aureus Staphylococcus aureus 6 Staphylococcusepidermidis Staphylococcus epidermidis 7 Staphylococcus lugdunensisStaphylococcus lugdunensis 8 Enterococcus faecalis Enterococcus faecalis9 Enterococcus faecium Enterococcus faecium 10 Bacillus subtilis groupBacillus amyloliquefaciens Bacillus atrophaeus Bacillus licheniformisBacillus subtilis 11 Bacillus cereus group Bacillus anthracis Bacilluscereus Bacillus thuringiensis 12 Micrococcus M. yunnanensis M.alkanovora M. aquilus M. endophyticus M. flavus M. indicus M. leuteus M.thailandius 13 Corynebacterium Corynebacterium jeikeium Corynebacteriumurealyticum Corynebacterium diphtheriae Corynebacterium ulceransCorynebacterium striatum And many more 14 Listeria Listeria innocuaListeria ivanovii Listeria seeligeri Listeria welshimeri 15 Listeriamonocytogenes L. monocytogenes 16 Lactobacillus Lactobacillus caseiLactobacillus paracasei Lactobacillus rhamnosus 17 Propionibacteriumacnes P. acnes 18 Enterococcus Enterococcus avium Enterococcuscasseliflavus Enterococcus faecalis Enterococcus faecium Enterococcusgallinarum Enterococcus hirae Enterococcus raffinosus Enterococcussaccharolyticus 19 Streptococcus Streptococcus agalactiae{circumflexover ( )} Streptococcus constellatus{circumflex over ( )} Streptococcusintermedius{circumflex over ( )} Streptococcus anginosus{circumflex over( )} Streptococcus bovis Streptococcus criceti Streptococcusdysgalactiae Streptococcus dysgalactiae subsp dysgalactiae Streptococcusdysgalactiae subsp equisimilis Streptococcus equi Streptococcus equinusStreptococcus gallolyricus Streptococcus gallolyricus pasteurianusStreptococcus gordonii Streptococcus infantarius Streptococcus infantisStreptococcus mitis Streptococcus mutans Streptococcus oralisStreptococcus parasanguinis Streptococcus peroris Streptococcuspneumoniae{circumflex over ( )} Streptococcus pyogenes{circumflex over( )} Streptococcus salivarius Streptococcus sanguinis Streptococcusthoraltensis 20 Staphylococcus Staphylococcus arlettae Staphylococcusaureus{circumflex over ( )} Staphylococcus auriculari Staphylococcuscapitis Staphylococcus caprae Staphylococcus carnosus Staphylococcuschromogenes Staphylococcus cohnii Staphylococcus epidermidis{circumflexover ( )} Staphylococcus gallinarum Staphylococcus haemolyticusStaphylococcus hominis Staphylococcus hominis subsp. novobiosepticusStaphylococcus hyicus Staphylococcus intermedius Staphylococcus lentusStaphylococcus lugdunensis{circumflex over ( )} Staphylococcus muscaeStaphylococcus pasteuri Staphylococcus pettenkoferi Staphylococcuspseudintermedius Staphylococcus saccharolyticus Staphylococcussaprophyticus Staphylococcus schleiferi Staphylococcus sciuriStaphylococcus simulans Staphylococcus vitulinus Staphylococcus warneriStaphylococcus xylosus 21 mecA Staphylococcus aureus (mecA)Staphylococcus epidermidis (mecA) 22 mecC Staphylococcus aureus (mecC)Staphylococcus epidermidis (mecC) 23 vanA Enterococcus faecalis (vanA)Enterococcus faecium (vanA) 24 vanB Enterococcus faecalis (vanB)Enterococcus faecium (vanB) 25 Pan Candida Candida albicans Candidaglabrata Candida krusei Candida parapsilosis 26 Pan Gram-NegativeEnterobacteriaceae Acinetobacter Pseudomonas BacteroidesStenotrophomonas {circumflex over ( )}identified in the species or groupcall

In a preferred embodiment the Pan Gram-negative target in the BCID-GPpanel can identify about 10 species of gram-negative bacteria. In apreferred embodiment the Pan Gram-negative target in the BCID-GP panelcan identify at least 5, at least 10, at least 15, at least 20, at least30, at least 40 at least 50 at least 60, at least 70, at least 80, atleast 90, at least 100 or more species of gram-negative bacteria. In apreferred embodiment the Pan Gram-negative target in the BCID-GP panelcan identify 30-100 species of gram-negative bacteria. In a preferredembodiment the Pan Gram-negative target in the BCID-GP panel canidentify about 10%, about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 80% or more species of gram-negative bacteria.

The BCID-GP oligonucleotides capable of binding a sequence of abacterial pathogen which enable discrimination between gram-positivespecies or genus were not designed to avoid or reduce detection ofbackground contamination. It was a surprising and unexpected result thatreducing cycling from 40 to 37, and in some cases from 40 to 35, and insome cases from 40 to 30 was sufficient to distinguish betweenbackground contamination, gram-positive bacteria species or genusinfection, non-species gram-negative bacteria and non-species fungalinfection.

The BCID-GP assay can be further understood by the following numberedparagraphs:

Paragraph 1. An in vitro method for the detection and/or identificationof a first human pathogen and/or genetic material thereof comprisingsubjecting a sample to a single multiplex polymerase chain reaction(PCR), wherein said method comprises amplification of PCR products underconditions appropriate for the substantial reduction or elimination inelectrochemical detection of contaminating pathogen and/or geneticmaterial present in the sample.

Paragraph 2. The method of Paragraph 1, wherein the first human pathogencomprises a gram-positive bacteria or a plurality of gram-positivebacteria.

Paragraph 3. The method of Paragraph 2, wherein the gram-positivebacteria is selected from the group consisting of Bacillus cereus group,Staphylococcus epidermidis, Bacillus subtilis group, Staphylococcuslugdunensis, Corynebacterium spp., Streptococcus, Enterococcus,Streptococcus agalactiae, Enterococcus faecalis, Streptococcus anginosusgroup, Enterococcus faecium, Streptococcus pneumonia, Lactobacillus,Streptococcus pyogenes, Listeria and combinations thereof.

Paragraph 4. The method of Paragraph 2, wherein the gram-positivebacteria is Streptococcus, Staphylococcus or Enterococcus faecalis.

Paragraph 5. The method of any preceding paragraph, wherein the methodcan further detect a second human pathogen if present in the sample.

Paragraph 6. The method of Paragraph 5, wherein the second humanpathogen is gram-positive bacteria, gram-negative bacteria, fungi, aplurality of gram-positive bacteria, a plurality of gram-negativebacteria, a plurality of fungi, or combinations thereof.

Paragraph 7. The method of Paragraph 6, wherein the gram-negativebacteria is selected from the group comprising Escherichia coli,Pseudomonas aeruginosa, Proteus mirabilis and combinations thereof.

Paragraph 8. The method of Paragraph 6, wherein the fungi are selectedfrom the group comprising Candida albicans, Candida glabrata, Candidakrusei, Candida parapsilosis and combinations thereof.

Paragraph 9. The method of any preceding paragraph, wherein the methodcan further detect an antimicrobial resistance gene.

Paragraph 10. The method of Paragraph 9, wherein the antimicrobialresistance gene is selected from the group consisting of mecA, mecC,vanA, and vanB.

Paragraph 11. The method of Paragraph 6, wherein the species of thegram-negative bacterial pathogen can be identified by subjecting thesample to a second single multiplex PCR, wherein said method comprisesamplification of PCR products under conditions appropriate for thesubstantial reduction or elimination in electrochemical detection ofcontaminating pathogen and/or genetic material present in the sample andwherein the gram-negative bacteria is selected from the group consistingof Acinetobacter baumannii, Klebsiella pneumoniae, Bacteroides fragilis,Morganella morganii, Citrobacter, Neisseria meningitides, Cronobactersakazakii, Proteus, Enterobacter cloacae complex, Proteus mirabilis,Enterobacter (non-cloacae complex), Pseudomonas aeruginosa, Escherichiacoli, Salmonella, Fusobacterium necrophorum, Serratia, Fusobacteriumnucleatum, Serratia marcescens, Haemophilus influenza, Stenotrophomonasmaltophilia, Klebsiella oxytoca and combinations thereof.

Paragraph 12. The method of Paragraph 6, wherein the species of fungipathogen can be identified by subjecting the sample to a second singlemultiplex PCR, wherein said method comprises amplification of PCRproducts under conditions appropriate for the substantial reduction orelimination in electrochemical detection of contaminating pathogenand/or genetic material present in the sample and wherein the fungi areselected from the group consisting of Candida albicans, Candidadubliniensis, Candida famata, Candida glabrata, Candida guilliermondii,Candida kefyr, Candida lusitaniae, Candida krusei, Candida parapsilosis,Candida tropicalis, Cryptococcus gattii, Cryptococcus neoformans,Fusarium, Malassezia furfur, Rhodotorula, Trichosporon and combinationsthereof.

Paragraph 13. The method of any preceding paragraph, wherein thedetection method is electrochemical detection.

Paragraph 14. The method of any preceding paragraph, whereincontaminating organisms from a blood draw are identified.

Paragraph 15. The method of paragraph 14, wherein the contaminatingorganisms are selected from the group comprising Propionibacteriumacnes, Staphylococcus epidermidis, Micrococcus, Lactobacillus orCorynebacterium.

Paragraph 16. An in vitro method for the detection and/or identificationof a human pathogen and/or genetic material thereof comprisingsubjecting a sample to a single multiplex polymerase chain reaction(PCR), wherein said method comprises amplification of PCR products underconditions appropriate for the substantial reduction or elimination inelectrochemical detection of contaminating pathogen and/or geneticmaterial present in the sample wherein the human pathogen isgram-positive bacteria, gram-negative bacteria, fungi or combinationsthereof.

Paragraph 17. A microfluidic device for the detection and/oridentification of a human pathogen and/or genetic material thereofcomprising: a mixture of oligonucleotides and reagents for carrying outa single nucleic acid amplification reaction capable of distinguishingbetween clinically relevant amplification and amplification from othersources such as from background contamination.

Paragraph 18. The microfluidic device of paragraph 17, wherein theclinically relevant amplification is a first human pathogen.

Paragraph 19. The microfluidic device of paragraph 18, wherein the firsthuman pathogen comprises a gram-positive bacteria or a plurality ofgram-positive bacteria.

Paragraph 20. The microfluidic device of Paragraph 19, wherein thegram-positive bacteria is selected from the group consisting of Bacilluscereus group, Staphylococcus epidermidis, Bacillus subtilis group,Staphylococcus lugdunensis, Corynebacterium spp., Streptococcus,Enterococcus, Streptococcus agalactiae, Enterococcus faecalis,Streptococcus anginosus group, Enterococcus faecium, Streptococcuspneumonia, Lactobacillus, Streptococcus pyogenes, Listeria andcombinations thereof.

Paragraph 21. The microfluidic device of any preceding Paragraph,wherein the method can further detect a second human pathogen if presentin the sample.

Paragraph 22. The microfluidic device of Paragraph 21, wherein thesecond human pathogen is gram-positive bacteria, gram-negative bacteria,fungi, a plurality of gram-negative bacteria, a plurality of fungi, orcombinations thereof.

Paragraph 23. The microfluidic device of Paragraph 22, wherein thegram-negative bacteria is selected from the group comprising Escherichiacoli, Pseudomonas aeruginosa, Proteus mirabilis and combinationsthereof.

Paragraph 24. The microfluidic device of Paragraph 22, wherein the fungiare selected from the group comprising Candida albicans, Candidaglabrata, Candida krusei, Candida parapsilosis and combinations thereof.

Paragraph 25. The microfluidic device of any preceding Paragraph,wherein the method can further detect an antimicrobial resistance gene.

Paragraph 24. The microfluidic device of Paragraph 23, wherein theantimicrobial resistance gene is selected from the group consisting ofmecA, mecC, vanA, and vanB.

Paragraph 25. The microfluidic device of any preceding paragraph,wherein contaminating organisms from a blood draw are identified.

Paragraph 26. The microfluidic device of paragraph 25, wherein thecontaminating organisms are selected from the group comprisingPropionibacterium acnes, Staphylococcus epidermidis, Micrococcus,Lactobacillus or Corynebacterium.

Paragraph 27. The microfluidic device of Paragraph 22, wherein thespecies of the gram-negative bacterial pathogen can be identified bysubjecting the sample to a single multiplex PCR, wherein said methodcomprises amplification of PCR products under conditions appropriate forthe substantial reduction or elimination in electrochemical detection ofcontaminating pathogen and/or genetic material present in the sample andwherein the gram-negative bacteria is selected from the group consistingof Acinetobacter baumannii, Klebsiella pneumoniae, Bacteroides fragilis,Morganella morganii, Citrobacter, Neisseria meningitides, Cronobactersakazakii, Proteus, Enterobacter cloacae complex, Proteus mirabilis,Enterobacter (non-cloacae complex), Pseudomonas aeruginosa, Escherichiacoli, Salmonella, Fusobacterium necrophorum, Serratia, Fusobacteriumnucleatum, Serratia marcescens, Haemophilus influenza, Stenotrophomonasmaltophilia, Klebsiella oxytoca and combinations thereof.

Paragraph 28. The microfluidic device of Paragraph 22, wherein thespecies of fungi pathogen can be identified by subjecting the sample toa single multiplex PCR, wherein said method comprises amplification ofPCR products under conditions appropriate for the substantial reductionor elimination in electrochemical detection of contaminating pathogenand/or genetic material present in the sample and wherein the fungi areselected from the group consisting of Candida albicans, Candidadubliniensis, Candida famata, Candida glabrata, Candida guilliermondii,Candida kefyr, Candida lusitaniae, Candida krusei, Candida parapsilosis,Candida tropicalis, Cryptococcus gattii, Cryptococcus neoformans,Fusarium, Malassezia furfur, Rhodotorula, Trichosporon and combinationsthereof.

Paragraph 29. The microfluidic device of any preceding Paragraph,wherein the detection method is electrochemical detection.

Paragraph 30. A microfluidic device for the detection and/oridentification of a human pathogen and/or genetic material thereofcomprising: a mixture of oligonucleotides and reagents for carrying outa single nucleic acid amplification reaction capable of distinguishingbetween clinically relevant amplification and amplification from othersources such as from background contamination wherein the clinicallyrelevant amplification is from a gram-positive bacteria, gram-negativebacteria, fungi or combinations thereof.

Gram-Negative

The BCID-GN Panel is a fully automated, qualitative, nucleic acid,multiplex in vitro diagnostic test for simultaneous detection andidentification of multiple potentially pathogenic gram-negativebacterial organisms and select determinants of antimicrobial resistancein positive blood culture. The test also detects but does notdifferentiate gram-positive bacteria and several pathogenic Candidaspecies. The test is able to detect 21 bacterial targets and 6resistance genes, as well as multiple Candida species from a singlecartridge (single PCR run) and most major gram-positive organisms, alsoas on a single cartridge (single PCR run).

The following bacterial organisms are identified using the BCID-GNPanel: Acinetobacter baumannii, Klebsiella pneumoniae, Bacteroidesfragilis, Morganella morganii, Citrobacter, Neisseria meningitides,Cronobacter sakazakii, Proteus, Enterobacter cloacae complex, Proteusmirabilis, Enterobacter (non-cloacae complex), Pseudomonas aeruginosa,Escherichia coli, Salmonella, Fusobacterium necrophorum, Serratia,Fusobacterium nucleatum, Serratia marcescens, Haemophilus influenza,Stenotrophomonas maltophilia, Klebsiella oxytoca. The followingAntimicrobial Resistance Markers are identified using the BCID-GN Panel:CTX-M, NDM, IMP, OXA, KPC, VIM. The following Pan Targets are identifiedusing the BCID-GN Panel: Pan Candida (Candida albicans, Candidaglabrata, Candida krusei, Candida parapsilosis) See FIG. 18; PanGram-Positive (S. anginosus group, Enterococcus, Staphylococcus,Streptococcus, Bacillus subtilis group, Bacillus cereus group,Enterococcus faecalis) See FIG. 18. So for the Pan-Gram-positive call inthe BCID-GN panel, a co-infection is detected by its species butidentified by the type, gram-positive.

Table 2 below shows that reported target call and the target speciesdetected. Stated another way, some species are detected by the BCID-GNpanel (“Targets detected” in Table 2 below) but not identified by thespecies in the call (report); instead, the call/report identifies thegenus. Some species are detected by the BCID-GN panel (“Targetsdetected” in Table 2 below) and identified by the species in the call(report). Some organisms can generate both the genus and species call.

TABLE 2 Gram-Negative Analytes Detected by the BCID-GN Panel ReportedTarget Targets Detected Acinetobacter baumannii Acinetobacter baumanniiBacteroides fragilis Bacteroides fragilis Citrobacter Citrobacter brakiiCitrobacter fruendii Citrobacter koseri Critrobacter youngae Citrobacterfreundii/brakii Citrobacter freundii Citrobacter brakii Cronobactersakazakii Cronobacter sakazakii Enterobacter (not cloacae Enterobacteraerogenes complex) Enterobacter amnigenus Enterobacter gergoviae (detectwith amnigenus assay) Enterobacter cloacae complex Enterobacter asburiaeEnterobacter cloacae Enterobacter hormaechei Escherichia coliEscherichia coli Fusobacterium (not necrophorum) Fusobacterium nucleatumFusobacterium russii Fusobacterium varium Fusobacterium periodonticumFusobacterium necrophorum Fusobacterium necrophoum Haemophilusinfluenzae Haemophilus influenza Klebsiella oxytoca Klebsiella oxytocaKlebsiella pneumoniae Klebsiella pneumoniae Klebsiella variicolaMorganella morganii Morganella morganii Neisseria meningitidis Neisseriameningitidis Pantoea agglomerans Pantoea agglomerans PrevotellaPrevotella bivia Prevotella buccae Prevotella buccalis Prevotellacorporis Prevotella dentalis Prevotella denticola Prevotella disiensPrevotella intermedia Prevotella oralis Prevotella oris Prevotellaveroralis Proteus Proteus hauseri Proteus mirabilis Proteus penneriProteus vulgaris Proteus mirabilis Proteus mirabilis Pseudomonasaeruginosa Pseudomonas aeruginosa Pseudomonas Pseudomonas aeruginosaPseudomonas oryzihabitans Pseudomonas alcaligenes Pseudomonasfluorescens Pseudomonas mendocina Pseudomonas pseudoalcaligenesPseudomonas putida Pseudomonas stutzeri Salmonella Salmonella bongoriSalmonella bongori Salmonella enterica subsp arizonae Salmonellaenterica subsp diarizonae Salmonella enterica subsp enterica serovarAbaetetuba Salmonella enterica subsp enterica serovar Abony Salmonellaenterica subsp enterica serovar Typhimurium Serratia marcescens Serratiamarcescens Serratia Serratia ficaria Serratia fonticola Serratiagrimesii Serratia liquefaciens Serratia plymuthica Serratia rubidaeaSerretia odorifera Stenotrophomonas maltophilia Stenotrophomonasmaltophilia CTX-M CTX-1 CTX-2 CTX-8 CTX-9 CTX-25 IMP IMP-1 IMP-18 IMP-33IMP-5 KPC KPC NDM NDM OXA OXA-23 OXA-48 VIM VIM

In a preferred embodiment the Pan Gram-positive target in the BCID-GNpanel can identify about 15 species of gram-positive bacteria. In apreferred embodiment the Pan Gram-positive target in the BCID-GN panelcan identify at least 10, at least 15, at least 20, at least 30, atleast 40 at least 50 at least 60, at least 70, at least 80, at least 90,at least 100 or more species of gram-positive bacteria. In a preferredembodiment the Pan Gram-positive target in the BCID-GN panel canidentify 30-100 species of gram-positive bacteria. In a preferredembodiment the Pan Gram-positive target in the BCID-GN panel canidentify about 10%, about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 80% or more species of gram-positive bacteria.

The BCID-GN oligonucleotides capable of binding a sequence of abacterial pathogen which enable discrimination between gram-negativespecies or genus were not designed to avoid or reduce detection ofbackground contamination. It was a surprising and unexpected result thatdetuning the assay i.e., by merely reducing the PCR cycling, wassufficient to distinguish between background contamination,gram-negative bacteria species or genus infection, non-speciesgram-positive bacteria and non-species fungal infection.

The BCID-GN Panel contains targets designed to detect a broad range oforganisms with a potentially misleading Gram stain result or organismsthat may be missed by Gram staining altogether for example in the caseof co-infections. These include a Pan Gram-Positive assay as well as aPan Candida assay, both of which may provide data to facilitate thecorrect testing algorithm. As such, the present disclosure relates tomethods and systems for a) distinguishing between backgroundcontamination and gram-negative bacterial infection; b) distinguishingbetween gram-negative bacterial species infection; c) distinguishingbetween some gram-negative bacterial species and some gram-negativegenus infection(s); and d) detecting but not identifying gram-positivebacterial species or genus infection and fungal species infection. Thepresent disclosure further relates to methods and systems foridentifying a pathogen that is likely a contamination from the blooddraw.

Gram-negative bacteria are a common cause of bacteremia, being isolatedfrom over 60% of positive blood cultures throughout the world.Antimicrobial resistance is common among gram-negative organisms, andmulti-drug resistance is increasingly common in many species. Wheninvolved in bacteremia, the species belonging to this group havemortality rates ranging from 20% to over 90% in some populations.

The BCID-GN assay can be further understood by the following numberedparagraphs:

Paragraph 1. An in vitro method for the detection and/or identificationof a first human gram-negative bacteria pathogen and/or genetic materialthereof comprising subjecting a sample to a single multiplex polymerasechain reaction (PCR), wherein said method comprises amplification of PCRproducts under conditions appropriate for the substantial reduction orelimination in electrochemical detection of contaminating pathogenand/or genetic material present in the sample.

Paragraph 2. The method of Paragraph 1, wherein the first human pathogencomprises a gram-negative bacteria or a plurality of gram-negativebacteria.

Paragraph 3. The method of Paragraph 2, wherein the gram-negativebacteria is selected from the group consisting of Acinetobacterbaumannii, Klebsiella pneumoniae, Bacteroides fragilis, Morganellamorganii, Citrobacter, Neisseria meningitides, Cronobacter sakazakii,Proteus, Enterobacter cloacae complex, Proteus mirabilis, Enterobacter(non-cloacae complex), Pseudomonas aeruginosa, Escherichia coli,Salmonella, Fusobacterium necrophorum, Serratia, Fusobacteriumnucleatum, Serratia marcescens, Haemophilus influenza, Stenotrophomonasmaltophilia, Klebsiella oxytoca and combinations thereof.

Paragraph 4. The method of any preceding paragraph, wherein the methodcan further detect a second human pathogen if present in the sample.

Paragraph 5. The method of Paragraph 4, wherein the second humanpathogen is gram-negative bacteria, gram-positive bacteria, fungi, aplurality of gram-negative bacteria, a plurality of gram-positivebacteria, a plurality of fungi, or combinations thereof.

Paragraph 6. The method of Paragraph 5, wherein the gram-positivebacteria is selected from the group comprising Staphylococcus,Streptococcus, Bacillus subtilis group, Bacillus cereus groupEnterococcus, Proteus mirabilis, Acinetobater baumannii, Serratia,Citrobacter, Enterococcus faecalis, Neisseria meningitides, Morganellamorganii, Klebsiella penumoniae, Haemophilus influenza or combinationsthereof.

Paragraph 7. The method of Paragraph 5, wherein the fungi are selectedfrom the group comprising Candida albicans, Candida glabrata, Candidakrusei, Candida parapsilosi or combinations thereof.

Paragraph 8. The method of any preceding paragraph, wherein the methodcan further detect an antimicrobial resistance gene.

Paragraph 9. The method of Paragraph 8, wherein the antimicrobialresistance gene is selected from the group consisting of CTX-M, NDM,IMPDXA, KPC, VIM or combinations thereof.

Paragraph 10. The method of Paragraph 5, wherein the species of thegram-positive bacterial pathogen can be identified by subjecting thesample to a single multiplex PCR, wherein said method comprisesamplification of PCR products under conditions appropriate for thesubstantial reduction or elimination in electrochemical detection ofcontaminating pathogen and/or genetic material present in the sample andwherein the gram-positive bacteria is selected from the group consistingof Bacillus cereus group, Staphylococcus epidermidis, Bacillus subtilisgroup, Staphylococcus lugdunensis, Corynebacterium spp., Streptococcus,Enterococcus, Streptococcus agalactiae, Enterococcus faecalis,Streptococcus anginosus group, Enterococcus faecium, Streptococcuspneumonia, Lactobacillus, Streptococcus pyogenes, Listeria, andcombinations thereof.

Paragraph 11. The method of Paragraph 5, wherein the species of fungipathogen can be identified by subjecting the sample to a singlemultiplex PCR, wherein said method comprises amplification of PCRproducts under conditions appropriate for the substantial reduction orelimination in electrochemical detection of contaminating pathogenand/or genetic material present in the sample and wherein the fungi areselected from the group consisting of Candida albicans, Candidadubliniensis, Candida famata, Candida glabrata, Candida guilliermondii,Candida kefyr, Candida lusitaniae, Candida krusei, Candida parapsilosis,Candida tropicalis, Cryptococcus gattii, Cryptococcus neoformans,Fusarium, Malassezia furfur, Rhodotorula, Trichosporon and combinationsthereof.

Paragraph 12. The method of any preceding paragraph, wherein thedetection method is electrochemical detection.

Paragraph 13. The method of any preceding paragraph, wherein if four ormore human pathogens are detected, the sample is subject to a new singlePCR run.

Paragraph 14. A microfluidic device for the detection and/oridentification of a human pathogen and/or genetic material thereofcomprising: a mixture of oligonucleotides and reagents for carrying outa single nucleic acid amplification reaction capable of distinguishingbetween clinically relevant amplification and amplification from othersources such as from background contamination.

Paragraph 15. The microfluidic device of paragraph 14, wherein theclinically relevant amplification is a first human pathogen.

Paragraph 16. The microfluidic device of paragraph 15, wherein the firsthuman pathogen comprises a gram-negative bacteria or a plurality ofgram-negative bacteria.

Paragraph 17. The microfluidic device of Paragraph 16, wherein thegram-negative bacteria is selected from the group consisting ofAcinetobacter baumannii, Klebsiella pneumoniae, Bacteroides fragilis,Morganella morganii, Citrobacter, Neisseria meningitides, Cronobactersakazakii, Proteus, Enterobacter cloacae complex, Proteus mirabilis,Enterobacter (non-cloacae complex), Pseudomonas aeruginosa, Escherichiacoli, Salmonella, Fusobacterium necrophorum, Serratia, Fusobacteriumnucleatum, Serratia marcescens, Haemophilus influenza, Stenotrophomonasmaltophilia, Klebsiella oxytoca and combinations thereof.

Paragraph 18. The microfluidic device of Paragraph 16, wherein thegram-negative bacteria is selected from the group consisting ofEscherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Proteus,and combinations thereof.

Paragraph 19. The microfluidic device of any preceding paragraph,wherein the device can further detect a second human pathogen if presentin the sample.

Paragraph 20. The microfluidic device of paragraph 18, wherein thesecond human pathogen is gram-negative bacteria, gram-positive bacteria,fungi, a plurality of gram-negative bacteria, a plurality ofgram-positive bacteria, a plurality of fungi, or combinations thereof.

Paragraph 21. The microfluidic device of Paragraph 19, wherein thegram-positive bacteria is selected from the group comprising Bacilluscereus group, Staphylococcus epidermidis, Bacillus subtilis group,Staphylococcus lugdunensis, Corynebacterium spp., Streptococcus,Enterococcus, Streptococcus agalactiae, Enterococcus faecalis,Streptococcus anginosus group, Enterococcus faecium, Streptococcuspneumonia, Lactobacillus, Streptococcus pyogenes, Listeria, andcombinations thereof.

Paragraph 22. The microfluidic device of Paragraph 19, wherein the fungiis selected from the group comprising Candida albicans, Candidadubliniensis, Candida famata, Candida glabrata, Candida guilliermondii,Candida kefyr, Candida lusitaniae, Candida krusei, Candida parapsilosis,Candida tropicalis, Cryptococcus gattii, Cryptococcus neoformans,Fusarium, Malassezia furfur, Rhodotorula, Trichosporon and combinationsthereof.

Paragraph 23. The microfluidic device of any preceding paragraph,wherein the method can further detect an antimicrobial resistance gene.

Paragraph 24. The microfluidic device of paragraph 23, wherein theantimicrobial resistance gene is selected from the group consisting ofCTX-M, NDM, IMPDXA, KPC, VIM or combinations thereof.

Paragraph 25. The microfluidic device of any preceding paragraph,wherein the detection method is electrochemical detection.

Fungal

The Blood Culture Identification Fungal Pathogen Panel (BCID-FP Panel)is a fully automated, qualitative, nucleic acid, multiplex in vitrodiagnostic test for simultaneous detection and identification ofmultiple potentially pathogenic fungal organisms in positive bloodculture. The BCID-FP Panel is performed directly on blood culturesamples identified as positive by a continuously monitoring bloodculture system that demonstrates the presence of organisms as confirmedby Gram stain.

The following fungal organisms are identified using the BCID-FP Panel:Candida auris, Candida albicans, Candida dubliniensis, Candida famata,Candida glabrata, Candida guilliermondii, Candida kefyr, Candidalusitaniae, Candida krusei, Candida parapsilosis, Candida tropicalis,Cryptococcus gattii, Cryptococcus neoformans, Fusarium, Malasseziafurfur, Rhodotorula, and Trichosporon. The fungal species detected bythe BCID-FP Panel are in FIG. 21. Specifically, Schizosaccharomycespombe, Malaessezia furfur, Candida albicans, and Candida auris get aspecies call. For the Fusarium call, the BCID-FP panel detects but doesnot identify the following species in the call (report): solani set,dimerum, proliferatum, moniliforme, verticillioides, oxysporum, andsacchari. For the Rhodotorula call, the BCID-FP panel detects but doesnot identify the following species in the call (report): mucilaginosa,and glutinis. For the Trichosporon call, the BCID-FP panel detects butdoes not identify the following species in the call (report): asteroid,coremiiforme and dermatis.

The BCID-FP oligonucleotides capable of binding a sequence of a fungalpathogen which enable discrimination between fungal species or genuswere not designed to avoid or reduce detection of backgroundcontamination. It was a surprising and unexpected result that merelycreating primer mismatches and in some cases using dual zone detectionwas sufficient to distinguish between background contamination andfungal species or genus infection. It was non-obvious to intentionallydecrease the sensitivity of the assay by intentionally introducingprimer miss-matches.

The present disclosure relates to methods and systems for a)distinguishing between background contamination and fungal infection.The present disclosure relates to methods and systems for a)distinguishing between background contamination, and detecting andidentifying the species or genus of fungal infection.

Invasive fungal infections are an increasingly common cause of sepsis incritically ill patients and are the source of significant morbidity andmortality. Of the fungi with the ability to cause severe sepsis, Candidaspecies are by far the most prevalent, accounting for between 8-10% ofall bloodstream infections in the US and 2-3% in Europe. Sepsis causedby invasive fungi is associated with mortality rates ranging from 15% tonearly 100% depending on the organism and underlying factors involved.

With increasing numbers of immunocompromised persons and increased useof implanted medical devices such as central venous catheters, theopportunity for infection with opportunistic pathogens is steadilyincreasing. This in combination with the fact that many fungi are partof the normal human skin, vaginal, and gastrointestinal flora and arecommonly found in the environment has resulted in a significant increasein fungal involvement in bloodstream infections.

The fungal primers, signal and capture probe sequences are below.Nucleotide sequences should have at least 80% sequence identitypreferably more than 85%, preferably more than 90%, preferably more than95% sequence identity, to the sequences provided herein.

TABLE 3 Fungal Forward And Reverse Primers and Sequences ID Nos. SpeciesForward Sequence Reverse Sequence Rhodotorula 1 SEQ. ID No. 1.SEQ. ID No. 5. XACTAGCACTACACGAGCA GGTAGTTCGGAGCGTGGAATAC CGGAAG CARhodotorula 2 SEQ. ID No. 2. SEQ. ID No. 6. XAGCACGGAAGTAGTAACCGGTCGTTTGGTACGTAGAATACC CATTAG A Trichosporon 1 SEQ. ID No. 3.SEQ. ID No. 7. ACTCTACACCGATTCTTCTA XATGTAATATGGATGCATTGGAA ACTTCA CTCGTrichosporon 2 SEQ. ID No. 4. SEQ. ID No. 8. ACACTTCACCGATTCTTCTAXATGTAATATGGATGCATTGGCA ACTTCA CTCG Trichosporon 3 SEQ. ID No. 9.XATATAATAAGGATGCATTGGA ATTCG

The BCID-FP assay can be further understood by the following numberedparagraphs:

Paragraph 1. An in vitro method for the detection and/or identificationof a first human fungal pathogen and/or genetic material thereofcomprising subjecting a sample to a single multiplex polymerase chainreaction (PCR), wherein said method comprises amplification of PCRproducts under conditions appropriate for the substantial reduction orelimination in electrochemical detection of contaminating pathogenand/or genetic material present in the sample.

Paragraph 2. The method of Paragraph 1, wherein the fungal pathogen isselected from the group consisting of Candida auris, Candida albicans,Candida dubliniensis, Candida famata, Candida glabrata, Candidaguilliermondii, Candida kefyr, Candida lusitaniae, Candida krusei,Candida parapsilosis, Candida tropicalis, Cryptococcus gattii,Cryptococcus neoformans, Fusarium, Malassezia furfur, Rhodotorula,Trichosporon and combinations thereof.

Paragraph 4. A microfluidic device for the detection and/oridentification of a human fungal pathogen and/or genetic materialthereof comprising: a mixture of oligonucleotides and reagents forcarrying out a single nucleic acid amplification reaction capable ofdistinguishing between clinically relevant fungal amplification andamplification from other sources such as from background contamination.

Paragraph 5. The microfluidic device of Paragraph 4, wherein the fugalpathogen is selected from the group consisting of Candida auris, Candidaalbicans, Candida dubliniensis, Candida famata, Candida glabrata,Candida guilliermondii, Candida kefyr, Candida lusitaniae, Candidakrusei, Candida parapsilosis, Candida tropicalis, Cryptococcus gattii,Cryptococcus neoformans, Fusarium, Malassezia furfur, Rhodotorula,Trichosporon and combinations thereof.

Paragraph 6. The microfluidic device of Paragraph 4, wherein the fugalpathogen is selected from the group consisting of Candida auris, C.parapsilosis, C. tropicalis, Rhodotorula, Trichosporon and combinationsthereof.

Paragraph 7. The microfluidic device of any preceding Paragraph, whereinthe detection method is electrochemical detection.

Two-Step Species Detection for Co-Infections

If a co-infection is of the same type as the infection (i.e., bothgram-positive, both gram-negative or both fungal), the method and systemcan identify the species of the co-infection based on the first singlePCR run. If a co-infection is of a different type as the infection(i.e., the infection is gram-positive but a pan gram-negative and/or panfungal co-infection is detected or the infection is gram-negative but apan gram-positive and/or pan fungal co-infection is detected), themethod and system can identify the genus of the fungal infection and thetype of the co-infection (gram-positive or negative) based on a secondsingle PCR run.

Gram staining is typically accurate, however, some organisms are knownto be gram variable, potentially producing misleading Gram stainresults. Additionally, inaccurate Gram stains have also been noted inthe instance of polymicrobial infections. The BCID-GP Panel includes twopan targets (Pan gram-negative and Pan Candida) designed to detect butnot identify organisms that may be missed by Gram stain. Likewise, theBCID-GN Panel includes two pan targets (Pan gram-positive and PanCandida) designed to detect but not identify organisms that may bemissed by Gram stain. If a pan target is identified in the BCID-GPPanel, then the BCID-GN and/or FN Panel can be run to identify thespecific species or genus of the infection. Likewise, if a pan target isidentified in the BCID-GN Panel, then the BCID-GP and/or FN Panel can berun to identify the specific species or genus of the infection.

In one embodiment, the method of the invention comprises the followingsteps: a) identify a first species or genus infection and aco-infection; b) identifying the species of the co-infection. In oneembodiment, the first infection is a gram-positive infection and theco-infection is a gram-negative infection or fungal infection. In oneembodiment, the first infection is a gram-negative infection and theco-infection is a gram-positive or fungal infection.

In one embodiment, the method of the invention comprises the followingsteps: a) providing a sample, b) bringing said sample into contact witha mixture of oligonucleotides and reagents for carrying out a nucleicacid amplification reaction, c) after DNA/neucleic acid extractioncarrying out a first single nucleic acid amplification reaction, d)obtaining a first result e) if four or more infections are present,obtaining a second result wherein obtaining a second result comprises f)providing the sample, g) bringing said sample into contact with amixture of oligonucleotides the same as the oligonucleotides used toobtained the first result and reagents for carrying out a nucleic acidamplification reaction, h) after DNA/neucleic acid extraction carryingout a second single nucleic acid amplification reaction, and i)obtaining a second result.

In one embodiment, the method of the invention comprises the followingsteps: a) obtaining a first result b) analyzing the first result for asecondary infection c) if a secondary infection is present obtaining asecond result wherein obtaining a second result comprises a) providing asample, b) bringing said sample into contact with a mixture ofoligonucleotides different from oligonucleotides used to obtained thefirst result and reagents for carrying out a nucleic acid amplificationreaction, c) carrying out a single nucleic acid amplification reaction,and d) obtaining a second result.

In one embodiment, the method of the invention comprises the followingsteps: a) obtaining a gram-stain result (b) selecting a panel based onthe gram stain result c) carry out a first single nucleic acidamplification reaction d) detecting the amplification products generatedas a result of said first single nucleic acid amplification reaction d)if the amplification products do not match the gram stain result, selecta second panel based on the results from the first single nucleic acidamplification reaction e) carry out a second single nucleic acidamplification reaction d) detect the amplification products generated asa result of said second single nucleic acid amplification reaction.

In embodiments, the second single nucleic acid amplification reactionprovides more specific species or genus identification than the firstsingle nucleic acid amplification reaction for the co-infection.

A method for identifying a fungal infection not identified by a gramstain comprising loading a sample suspected of having a bacterialinfection based on a gram stain into a first cartridge, amplifying thesample using a single nucleic acid amplification reaction, identifying apan-candida organism, and loading the sample into a second cartridgeamplifying the sample using a second single nucleic acid amplificationreaction and detecting the amplification products generated as a resultof said second single nucleic acid amplification reaction.

A method for screening a patient suspected of having a bacterialinfection comprising a) performing a first test for the presence of agram-positive bacterial infection; b) if the first test indicates agram-negative bacterial infection is present performing a second testfor the presence of a gram-negative bacterial infection wherein thefirst test and second test comprise amplifying the sample using a singlenucleic acid amplification reaction.

A method for screening a patient suspected of having a fungal infectioncomprising a) performing a first test for the presence of a bacterialinfection; b) if the first test indicates a fungal infection is presentperforming a second test for the presence of a fungal infection whereinthe first test and second test comprise amplifying the sample using asingle nucleic acid amplification reaction.

A method for identifying a plurality of organisms in a sample,comprising providing a first portion of a sample from a patient to afirst cartridge, bringing said first sample into contact with a mixtureof oligonucleotides and reagents for carrying out a single nucleic acidamplification reaction, determining if a second pathogen is present insaid first sample, providing a second portion of a sample from thepatient to a second cartridge, b) bringing said sample into contact witha mixture of oligonucleotides and reagents for carrying out a secondsingle nucleic acid amplification reaction, determining if a secondpathogen is present in said second sample.

The method of determining the existence of and identifying any one of upto a plurality of human pathogens in a sample, comprising the steps of:a) after DNA/neucleic acid extraction, performing a first detectionprocess comprising carrying out a single nucleic acid amplificationreaction, b) detecting and evaluating the amplification productsgenerated as a result of said first single nucleic acid amplificationreaction, c) after DNA/neucleic acid extraction, performing a seconddetection process comprising carrying out a single nucleic acidamplification reaction, and d) detecting and evaluating theamplification products generated as a result of said second detectionprocess, thereby identifying any one of up to a plurality of humanpathogens in the sample.

Amplification of Target Analytes

The BCID-GP, GN and FP panels can be run in a cartridge comprising abottom substrate. The bottom substrate (or printed circuit board) cancontain 1, 2, 3 or more amplification pathways or pads (called theAmplification Zone). These can be used for individual PCR reactions(e.g. one droplet is moved up one path and down another, etc.) or formultiplexing (e.g. eight different droplets can be moved up and downfour pathways).

As will be appreciated by those in the art, each PCR reaction canadditionally be multiplexed. That is, for target specific amplification,the use of multiple primer sets in a single PCR reaction can beunwieldy, and thus the invention allows multiple reactions to achievehigher levels of multiplexing. For example, for the evaluation of 21different target sequences (for example, in screening for fungalinfections), it may be desirable to run 3 different reactions of sevenprimer sets; e.g. a first PCR sample droplet (e.g. the bottom pathway)picks up the first set of 7 primer pairs (e.g. “Primer Mix A”), a seconddroplet picks up the second set of 7 primer pairs (“Primer Mix B”), anda third droplet picks up a third set (“Primer Mix C”). In someembodiments, the primers will be completely different in each set; inothers, redundancy and/or internal controls are built into the system byadding the same primer sets to different tracks. The multiplexingflexibility represents one of the key advantageous and distinguishingfeatures of the invention. The number of multiplexes can vary easilythrough software without the need to modify any physical components ofthe system.

In general, the amplification reactions (as more fully described below)for use in the present systems use sets of primers wherein one primer ofeach set has a blocked end that is impervious to standard exonucleases.That is, one strand of the double stranded amplicons that are generatedin the PCR reaction is removed so that the resulting single stranded DNAamplicon can hybridize to the single stranded capture probe. Thus, byrunning a first PCR reaction and then adding exonuclease, one strand ofthe double stranded amplicon is digested, leaving only the detectionstrand.

The use of heating zones perpendicular to the amplification pathway, asgenerally depicted in FIG. 15, allows the droplets to travel through theappropriate thermal zones. As shown in FIG. 15, four amplificationpathways are shown with three perpendicular thermal zones (in this case,the thermal elements are off chip Peltier heaters and show desiredtemperatures of about 95.5 C for denaturation (typically greater than90° C.) and about 65° C. (typically 60-70° C.) for annealing andextension. In this configuration, two-step amplification cycles can beperformed with more than one droplet in each PCR track, sometimesreferred to herein as “tandem amplification” or “bussing amplification”.For example, two droplets may be positioned in each PCR track and spacedin such a way that when one droplet is in the denaturation zone, theother is in one of combined annealing and extension zones, and viceversa. By shuttling the droplets in tandem back and forth between thedenaturation and annealing/extension zones, one can amplify both of themin the same amount of time it would normally take to amplify a singledroplet. In a four-track PCR configuration, this means that eightdroplets can be amplified simultaneously instead of four.

Detection of Amplification Products

The BCID-GP, GN and FP panels can be run on an automated nucleic acidtesting system including extraction, amplification, and detection,combining electrowetting and electrochemical detection. Electrochemicaldetection technology is based on the principles of competitive DNAhybridization and electrochemical detection, which is highly specificand is not based on fluorescent or optical detection.

Electrowetting, or digital microfluidics, uses electrical fields todirectly manipulate discrete droplets on the surface of ahydrophobically coated printed circuit board (PCB). Sample and reagentsare moved in a programmable fashion in the cartridge to complete allportions of the sample processing from nucleic acid extraction todetection.

A sample is loaded into the cartridge and the cartridge is placed intothe instrument. Nucleic acids are extracted and purified from thespecimen via magnetic solid phase extraction (i.e. the use of magneticbeads to pre-concentrate analytes or targets, then move (elute) thebeads containing the targets to a different location, where the targetsare released for post-elution events. PCR is used to createddouble-stranded cDNA which is treated with exonuclease to createsingle-stranded DNA in preparation for electrochemical detection.

The target amplicons are mixed with ferrocene-labeled signal probes thatare complementary to the specific targets on the panel. Target sequenceshybridize to the complementary signal probe and capture probes, whichare bound to gold-plated electrodes, as shown in FIG. 1. The presence ofeach target is determined by voltammetry which generates specificelectrical signals from the ferrocene-labeled signal probe.Specifically, FIG. 1 shows the hybridization complex. Target-specificcapture probes are bound to the gold electrodes in the microarray on thecartridge. The amplified target DNA hybridizes to the capture probe andto a complementary ferrocene-labeled signal probe. The electrochemicalanalysis determines the presence or absence of targets usingvoltammetry. The use of microfluidic systems in the electrochemicaldetection of target analytes is described in more detail in U.S. Pat.Nos. 9,557,295, 8,501,921, 6,600,026, 6,740,518 and U.S. applicationSer. No. 14/538,506 which are herein incorporated by reference in theirentirety.

Initial sample processing begins with blood draw, removal of blood fromthe tube at the lab, centrifugation, gram stain. Following gram stain,sample processing is summarized here: Step 1. Obtain sample after gramstain; Step 0. Load Sample; step 1. Combine Lysis Buffer with Sample(LRM), beads and Dispense Oil (Cartridge sub-assembly); step 2. CombineBinding Buffer with Sample (LRM) and Dispense Reconstitution Buffer(cartridge sub-assembly); step 3. Separate beads from sample beadmixture (LRM) and Rehydrate PCR reagent (cartridge sub-assembly); step4. Wash beads with Wash buffer (LRM) and Rehydrate PCR reagent(cartridge sub-assembly); step 5. Flush beads from LRM into cartridge;step 6. Final bead wash in cartridge sub-assembly and Quick Rinses(cartridge sub-assembly); step 7. Elute target analyte from beads; step8. Combine PCR reagent with elute target (analyte); step 9. Dispenseanalyte drops mix into PCR staging area; step 10. Rehydrate PCR primerscocktail with each analyte drop; step 11. Transfer eluted analyte tothermal-cycling PCR area in the cartridge; step 12. Convert RNA into DNAwith Reverse Transcriptase (optional step); step 13. Perform PCRcycling; step 14. Rehydrate exonuclease reagent; step 15. Combine PCRproducts with exonuclease reagent (ssDNA conversion); step 16.Exonuclease incubation and combine with Signal Probe cocktail(detection); step 17. Deliver PCR products and signal probe intoDetection area; step 18. Incubate in eSensor area with capture probebound to gold electrode; step 19. Scan and detect target analyte; step20. Eject cartridge.

The basic microfluidic platform used herein is based on systemsdeveloped by Advanced Liquid Logic (ALL, currently a subsidiary ofIllumina, Inc.), as more fully described in U.S. Patent app. no.20140194305 (which is incorporated by reference in its entirety). Ingeneral, these technologies rely on the formation of microdroplets andthe ability to independently transport, merge, mix and/or process thedroplets, using electrical control of surface tension (i.e.,electrowetting). In general, liquid samples are contained within amicrofluidic device between two parallel plates. One plate containsetched drive electrodes on its surface while the other plate containseither etched electrodes or a single, continuous plane electrode that isgrounded or set to a reference potential (“biplanar electrowetting”).Hydrophobic insulation covers the electrodes and an electric field isgenerated between electrodes on opposing plates. This electric fieldcreates a surface-tension gradient that causes a droplet overlapping theenergized electrode to move towards that electrode. In some embodiments,the active electrowetting electrodes may be adjacent and on the sameplane as the neighboring ground reference electrode, which is referredto as “coplanar electrowetting”). Through proper arrangement and controlof the electrodes, a droplet can be transported by successivelytransferring it between adjacent electrodes. The patterned electrodescan be arranged in a two dimensional array so as to allow transport of adroplet to any location covered by that array. The space surrounding thedroplets may be filled with a gas such as air or an immiscible fluidsuch as oil, with immiscible oils being preferred embodiments of thepresent invention. Indeed, the immiscible fliud may be a syntheticsilicone oil. This silicone oil is present throughout the system, i.e.,during amplification and detection.

Signal probes are used in the electrochemical detection of targetanalytes on the surface of a monolayer. QW56 or QW80 are ferrocenelabeled signal probes that can be prepared using routine DNA synthesistechniques essentially as described in commonly owned applicationPCT/US08/82666 (published as WO/2009/061941A2 and U.S. Pat. No.7,820,391 which are herein incorporated by reference in its entirety).In U.S. Application Ser. No. 14/218,61 (which is herein incorporated byreference in its entirety), FIG. 3A depicts QW 56 and FIG. 3B depictsWW80. N6 (a ferrocene labeled signal probe) is another label that can beused; its synthesis is described in commonly owned U.S. Pat. No.7,393,645 which is herein incorporated by reference in its entirety.

Capture probes are used in the electrochemical detection of targetanalytes on the surface of a monolayer. Specifically, capture bindingligands (called capture probes when the target analyte is a nucleicacid) anchor target analytes to the electrode surface and form an assaycomplex. The assay complex further comprises an electron transfer moiety(ETM), that is directly or indirectly attached to the target analyte.That is, the presence of the ETM near the electrode surface is dependenton the presence of the target analyte. Electron transfer between the ETMand the electrode is intiated using a variety of techniques as known bythose of skill in the art, and the output signals received andoptionally processed as further known by those of skill in the art.Thus, by detecting electron transfer, the presence or absence of thetarget analyte is determined.

In general, there are two basic detection mechanisms that may be used.In a preferred embodiment, detection of an ETM is based on electrontransfer through the stacked i-orbitals of double stranded nucleic acid.This basic mechanism is described in U.S. Pat. Nos. 5,591,578,5,770,369, 5,705,348, and PCT US97/20014 and is termed “mechanism-1”herein. Briefly, previous work has shown that electron transfer canproceed rapidly through the stacked i-orbitals of double strandednucleic acid, and significantly more slowly through single-strandednucleic acid. Accordingly, this can serve as the basis of an assay.Thus, by adding ETMs (either covalently to one of the strands ornon-covalently to the hybridization complex through the use ofhybridization indicators, described below) to a nucleic acid that isattached to a detection electrode via a conductive oligomer, electrontransfer between the ETM and the electrode, through the nucleic acid andconductive oligomer, may be detected.

Alternatively, the presence or absence of ETMs can be directly detectedon a surface of a monolayer. That is, the electrons from the ETMs neednot travel through the stacked π orbitals in order to generate a signal.As above, in this embodiment, the detection electrode preferablycomprises a self-assembled monolayer (SAM) that serves to shield theelectrode from redox-active species in the sample. In this embodiment,the presence of ETMs on the surface of a SAM, that has been formulatedto comprise slight “defects” (sometimes referred to herein as“microconduits”, “nanoconduits” or “electroconduits”) can be directlydetected. This basic idea is termed “mechanism-2” herein. Essentially,the electroconduits allow particular ETMs access to the surface. Withoutbeing bound by theory, it should be noted that the configuration of theelectroconduit depends in part on the ETM chosen. For example, the useof relatively hydrophobic ETMs allows the use of hydrophobicelectroconduit forming species, which effectively exclude hydrophilic orcharged ETMs. Similarly, the use of more hydrophilic or charged speciesin the SAM may serve to exclude hydrophobic ETMs. Thus, in eitherembodiment, an assay complex is formed that contains an ETM, which isthen detected using the detection electrode and the signal processingtechniques outlined herein.

Moreover, as specifically described in U.S. Pat. No. 6,740,518 which isherein incorporated by reference in its entirety, monitoring of theoutput signal at higher harmonic frequencies can be used to acheivehigher signal to noise ratios, to increase the detection limits oftarget analytes. For example, the ferrocene response reactsnon-linearly, producing a harmonic response in the signal above that inthe background; this harmonic signal from AC voltametry is most likelythe result of a harmonic distortion due to the nonlinear response of theelectrochemical cell; see Yap, J. of Electroanalytical Chem. 454:33(1998); hereby incorporated by reference. Thus, any techniques thatincrease this non-linearity are desirable. In a preferred embodiment,techniques are used to increase the higher harmonic signals; thus,frequency and phase-sensitive lock-in detection is performed at both thefundamental frequency of the applied waveform and also at multiples ofthe fundamental frequency (i.e. the higher harmonics). Since thebackground capacitance responds relatively linearly to AC signals (asine wave input AC voltage results in a relatively nondistorted sinewave output), very little upper harmonic current is produced in thebackground. This gives a dramatic increase in the signal to noise ratio.Thus, detection at the higher harmonic frequencies, particularly thethird, fourth and fifth harmonics (although the harmonics from second totenth or greater can also be used) is shown to result in dramaticsuppression of the background currents associated with non-Faradaicprocesses (like double layer charging) that can overwhelm the signalfrom the target molecules. In this way, the evaluation of the system athigher harmonic frequencies and phases can lead to significantimprovements in the detection limits and clarity of signal. Thus, in apreferred embodiment, one method of increasing the non-linear harmonicresponse is to increase or vary the amplitude of the AC perturbation,although this may also be used in monitoring the fundamental frequencyas well. Thus, the amplitude may be increased at high frequencies toincrease the rate of electron transfer through the system, resulting ingreater sensitivity. In addition, this may be used, for example, toinduce responses in slower systems such as those that do not possessoptimal spacing configurations

Electrode initialization is another signal processing method to acheivehigher signal to noise ratios, and to increase the detection limits oftarget analytes. In general, in any system, the observed signal is acombination of signal from the target analyte (sample signal) and signalfrom the background, or noise. Electrode initialization providesvariations in initiation signals (e.g. varying the “input”) that can beused to increase the signal, decrease the noise, or make the signal moreobvious or detectable in a background of noise. In an embodiment, theinput signal is AC/DC offset. In an embodiment, the AC frequency rangesfrom 90-1000 Hz. In an embodiment, the AC voltage ranges from −150 to880 mV rms. In an embodiment, electrode initialization is performed for0.5-5 seconds and then stopped as described in U.S. patent applicationSer. No. 14/218,615 which is herein infprorated by reference in itsentirety.

These techniques are generally described in U.S. application Ser. No.14/062,860 and U.S. Pat. Nos. 4,887,455; 5,591,578; 5,705,348;5,770,365; 5,807,701; 5,824,473; 5,882,497; 6,013,170; 6,013,459;6,033,601; 6,063,573; 6,090,933; 6,096,273; 6,180,064; 6,190,858;6,192,351; 6,221,583; 6,232,062; 6,236,951; 6,248,229; 6,264,825;6,265,155; 6,290,839; 6,361,958; 6,376,232; 6,431,016; 6,432,723;6,479,240; 6,495,323; 6,518,024; 6,541,617; 6,596,483; 6,600,026;6,602,400; 6,627,412; 6,642,046; 6,655,010; 6,686,150; 6,740,518;6,753,143; 6,761,816; 6,824,669; 6,833,267; 6,875,619; 6,942,771;6,951,759; 6,960,467; 6,977,151; 7,014,992; 7,018,523; 7,045,285;7,056,669; 7,087,148; 7,090,804; 7,125,668; 7,160,678; 7,172,897;7,267,939; 7,312,087; 7,381,525; 7,381,533; 7,384,749; 7,393,645;7,514,228; 7,534,331; 7,560,237; 7,566,534; 7,579,145; 7,582,419;7,595,153; 7,601,507; 7,655,129; 7,713,711; 7,759,073; 7,820,391;7,863,035; 7,935,481; 8,012,743; 8,114,661, 9,598,722, all of which areincorporated by reference in their entirety.

The automated nucleic acid testing system aka electrochemical detectionsystem described above includes a) an instrument bank comprising aplurality of biochip cartridge bays for insertion and analysis of abiochip cartridge, wherein each bay comprises: i) a top bay comprisingactuators for a liquid reagent module (LRM); and ii) a bottom baycomprising electrical connections for an electrowetting electrode gridand detection electrodes; and b) a base station comprising: i) a centralprocessing unit; and ii) a user interface comprising a touch screendisplay having a plurality of bay icons, each icon uniquelycorresponding to one of said plurality of bays. The sample-to-answersystem is generally described in U.S. patent application Ser. No.14/062,865, U.S. Pat. No. 9,598,722 and Provisional U.S. PatentApplication 62/396,449 all of which are incorporated by reference intheir entirety.

Identification, detection or reporting results occurs when amplifiedtarget DNA hybridizes to its complementary signal probe and captureprobes. Identification, detection or reporting results occurs whenamplified target DNA hybridizes to its complementary signal probe andcapture probes wherein the capture probe is bound to gold-platedelectrodes. Identification, detection or reporting results occurs when ahybridization complex forms between the target DNA and signal andcapture probes.

Detection of a target analyte can be further understood by the followingnumbered paragraphs:

Paragraph 1: A method for detecting the presence of a target analyte ina sample, the method comprising: a) providing an electrode comprising amonolayer and a capture binding ligand; b) initializing the electrode;c) hybridizing a probe to said target analyte to form an assay complex;and d) detecting the presence or absence of said target analyte whereinsaid detection comprises the substantial reduction in detection ofcontaminating pathogen and/or genetic material present in the sample.

Paragraph 2: A method of determining the presence of target analytes ina sample comprising: a) applying said sample to an array comprising aplurality of electrodes, wherein at least one electrode comprises anassay complex comprising: i) a capture binding ligand covalentlyattached to said electrode; ii) a target analyte; and iii) an electrontransfer moiety; b) applying an input waveform to said electrode togenerate an output waveform comprising at least one harmonic component,having a harmonic number greater than or equal to two; c) detecting saidoutput waveform at said electrode; d) analyzing said harmonic componentwith harmonic number greater than or equal to two to determine thepresence of said target analytes wherein said method comprises thesubstantial reduction in detection of contaminating pathogen and/orgenetic material present in the sample.

Paragraph 3: An immobilized capture probe carrier comprising: a firstcapture probe for detecting a species of pathogenic bacterium, the firstcapture probe being arranged on a solid phase carrier; and a secondcapture probe for detecting a genus of pathogenic fungi or type ofpathogenic gram-positive or pathogenic gram-negative bacteriumimmobilized at a position spaced from the first capture probe whereinthere is substantial reduction in detection of contaminating pathogenand/or genetic material present in the sample.

Paragraph 4: An signal probe carrier comprising: a first signal probebound to a ferrocene label for detecting a species of pathogenicbacterium; and a second signal probe for detecting a genus of pathogenicfungi or type of pathogenic gram-positive or pathogenic gram-negativebacterium wherein there is substantial reduction in detection ofcontaminating pathogen and/or genetic material present in the sample.

Paragraph 5: An in vitro method for the detection and/or identificationof a hybridization complex immobilized on a gold substrate comprising ahuman pathogen and/or genetic material thereof hybridized to a signalprobe with a ferrocene label and a capture probe comprising: subjectinga sample comprising or suspected of comprising a human pathogen and/orgenetic material thereof to a single multiplex polymerase chain reaction(PCR), wherein said method comprises amplification of PCR products underconditions appropriate for the substantial reduction in detection ofcontaminating pathogen and/or genetic material present in the sample.

Paragraph 6: A method for detecting the presence of a target analyte ina sample, the method comprising: a) subjecting the sample to a singlemultiplex polymerase chain reaction (PCR), wherein said method comprisesamplification of PCR products under conditions appropriate for thesubstantial reduction or elimination in electrochemical detection ofcontaminating pathogen and/or genetic material present in the sample andamplification of a species specific sequence of a gram-positivebacterial pathogen and not gram-negative bacteria or fungi; b) providingan electrode comprising a monolayer and a capture binding ligand; c)hybridizing said amplified target analyte to said capture binding ligandto form an assay complex; and d) detecting the presence or absence ofsaid target analyte wherein said detection comprises the substantialreduction in detection of contaminating pathogen and/or genetic materialpresent in the sample. In some embodiments, step a can further detect asecond human pathogen if present in the sample wherein the second humanpathogen is gram-positive bacteria, gram-negative bacteria, or fungi. Insome embodiment's species of the gram-negative bacterial pathogen orfungal pathogen can be identified by subjecting the sample to a secondsingle multiplex PCR, wherein said method comprises amplification of PCRproducts under conditions appropriate for the substantial reduction orelimination in electrochemical detection of contaminating pathogenand/or genetic material present in the sample.

Paragraph 7: A method for detecting the presence of a target analyte ina sample, the method comprising: a) subjecting the sample to a singlemultiplex polymerase chain reaction (PCR), wherein said method comprisesamplification of PCR products under conditions appropriate for thesubstantial reduction or elimination in electrochemical detection ofcontaminating pathogen and/or genetic material present in the sample andamplification of a species specific sequence of a gram-negativebacterial pathogen and not gram-positive bacteria or fungi; b) providingan electrode comprising a monolayer and a capture binding ligand; c)hybridizing said amplified target analyte to said capture binding ligandto form an assay complex; and d) detecting the presence or absence ofsaid target analyte wherein said detection comprises the substantialreduction in detection of contaminating pathogen and/or genetic materialpresent in the sample. In some embodiments, step a can further detect asecond human pathogen if present in the sample wherein the second humanpathogen is gram-positive bacteria, gram-negative bacteria, or fungi. Insome embodiments, step a can further detect a second human pathogen ifpresent in the sample by its genus or gram type. In some embodiment'sspecies of the gram-positive bacterial pathogen or fungal pathogen canbe identified by subjecting the sample to a second single multiplex PCR,wherein said method comprises amplification of PCR products underconditions appropriate for the substantial reduction or elimination inelectrochemical detection of contaminating pathogen and/or geneticmaterial present in the sample.

Paragraph 8: A microfluidic device for detecting a human pathogen and/orgenetic material thereof comprising: a housing, a liquid reagent modual,a top plate and a bottom substrate the bottom substrate comprising amixture of oligonucleotides and reagents for carrying out a singlenucleic acid amplification reaction capable of distinguishing betweencontaminating pathogen and/or genetic material present in the sample andinfectious pathogen and/or genetic material present in the sample andcapable of distinguishing between gram-negative bacterial species andidentifying by its genus a fungal co-infection or identifying by itstype a gram-positive co-infection.

Paragraph 9: An immobilized capture probe capable of capable ofdistinguishing between contaminating pathogen and/or genetic materialpresent in the sample and infectious pathogen and/or genetic materialpresent in the sample and capable of distinguishing betweengram-negative bacterial species and identifying by its genus a fungalco-infection or identifying by its type a gram-positive co-infection.

Paragraph 10: A signal probe capable of capable of distinguishingbetween contaminating pathogen and/or genetic material present in thesample and infectious pathogen and/or genetic material present in thesample and capable of distinguishing between gram-negative bacterialspecies and identifying by its genus a fungal co-infection oridentifying by its type a gram-positive co-infection.

Paragraph 11: An in vitro method for the detection and/or identificationof a human pathogen and/or genetic material thereof comprisingsubjecting a sample to a single multiplex polymerase chain reaction(PCR), wherein said method comprises amplification of PCR products underconditions appropriate for the substantial reduction or elimination indetection of contaminating pathogen and/or genetic material present inthe sample wherein the human pathogen is gram-positive bacteria,gram-negative bacteria, fungi or combinations thereof wherein the PCRproducts are cycled 30-35 times and are moved between heaters usingelectrowetting.

Paragraph 12: A method of treating a patient having or suspected ofhaving a bacterial infection comprising: obtaining a blood sample;subjecting the sample to a single multiplex polymerase chain reaction(PCR), wherein said method comprises amplification of PCR products underconditions appropriate for the substantial reduction or elimination inelectrochemical detection of contaminating pathogen and/or geneticmaterial present in the sample wherein the human pathogen isgram-positive bacteria, gram-negative bacteria, fungi or combinationsthereof; detecting the presence of a clinically relevant pathogen andtreating the patient based on detection.

Paragraph 13: A method of detecting a human pathogen in a sample, themethod comprising: obtaining a sample; detecting whether a humanpathogen is present by subjecting the sample to a single multiplexpolymerase chain reaction (PCR), wherein said method comprisesamplification of PCR products under conditions appropriate for thesubstantial reduction or elimination in electrochemical detection ofcontaminating pathogen and/or genetic material present in the samplewherein the human pathogen is gram-positive bacteria, gram-negativebacteria, fungi or combinations thereof.

Paragraph 14: A method of diagnosing a gram-positive bacterial infectionor gram-negative bacterial infection or fingal infection in a patient,said method comprising: obtaining a blood sample; subjecting the sampleto a single multiplex polymerase chain reaction (PCR), wherein saidmethod comprises amplification of PCR products under conditionsappropriate for the substantial reduction or elimination inelectrochemical detection of contaminating pathogen and/or geneticmaterial present in the sample; detecting whether a gram-positivebacteria or gram-negative bacterial infection or fingal infection ispresent by contacting the the PCR products with a signal and captureprobe and detecting binding between the PCR products and the signal andcapute probe; and diagnosing the patient with a gram-positive infectionwhen the presence of a gram-positive bacterial identified by its speciesor genus is detected or diagnosing the patient with a gram-negativeinfection when the presence of a gram-negative bacterial identified byits species or genus is detected or diagnosing the patient with a fungalinfection when the presence of a fungi is identified by its species orgenus is detected.

Paragraph 14: A method of diagnosing and treating a gram-positivebacterial infection or gram-negative bacterial infection or fingalinfection in a patient, said method comprising: obtaining a bloodsample; subjecting the sample to a single multiplex polymerase chainreaction (PCR), wherein said method comprises amplification of PCRproducts under conditions appropriate for the substantial reduction orelimination in electrochemical detection of contaminating pathogenand/or genetic material present in the sample; detecting whether agram-positive bacteria or gram-negative bacterial infection or fingalinfection is present by contacting the the PCR products with a signaland capture probe and detecting binding between the PCR products and thesignal and capute probe; and administering an effective amount ofantibiotic or anti-fungal to the diagnosed patient.

Sample-To-Answer System

The sample-to-answer system combines an automated nucleic acid testingsystem with communication capabilities to streamline the diagnosticworkflow from physician order entry to the release of the final reportwith accurate, actionable test results.

The sample-to-answer system is designed to reduce avoidable medicalerrors. Preventable medical errors are now the third leading cause ofdeath in the United States at more than 250,000 per year. See Martin AMakary, Michael Daniel. Medical error—the third leading cause of deathin the US. BMJ, 2016. Automating information transfer has been shown tobe effective in reducing many common errors, including patient identitychecking and result transcription. The sample-to-answer system isuniquely designed with patient safety features to help address thischallenge in the lab.

Bi-Directional LIS

The sample-to-answer system includes a bi-directional LIS (also referredto as a communication system, an LIS communication system,bi-directional LIS communication system and the like) to automate andaccelerate order entry and results reporting. See FIG. 2 for a schematicof the bi-directional LIS reporting. Specifically, when a sample iscollected, the physician creates a test order called a physician testorder. The physician test order allows the physician to specify a samplestability time (the time the sample is stable before results could beaffected). The physician test order will further allow the physician tospecify the time that a physician test order should remain on thephysician test order list before processing.

Physician→Hospital LIS

After the physician test order is generated, the physician test order issent to the hospital's laboratory information system (LIS), a computersoftware that processes, stores and manages data from all stages ofmedical processes and tests. The physician test order is accepted by theHospital LIS and a pending test order (PTO) is created once the patientsample is received and accessioned by the lab into the hospital LIS.

Hospital LIS→LIS Interchange

The hospital's LIS sends the PTO to an LIS interchange which convertsthe PTO request from an HL7 or ASTM format to a CSV format and the PTOis now referred to as a test order or an interchange order or formattedtest order and the like. HL7 and ASTM are a set of standards used in thetransfer of information between clinical instruments and LaboratoryInformation Systems. In this way, the sample-to-answer system is able tocommunicate with any hospital LIS because it is driven by multiplestandard messaging protocols such as HL7 and ASTM. In this way, if thehospital's LIS system is updated the LIS interchange can be remotelyupdated (an update on the clinical instrument is not required).

The sample-to-answer system further supports a “flat file format” i.e.non-standard file support for laboratories without automated interfaces(HL7 or ASTM). As such, tests can be imported and/or exported manuallyin a text format, CSV, TXT or XML formats. Automatic results can bereleased in XML format to a shared network location.

When the LIS interchange receives the PTO and reformats it to a testorder, the test order is auto published with information associated withthe PTO/sample such as patient identification, accession number, testordered (BCID-GP, BCID-GN, BCID-FP etc), patient type (e.g. pediatric,intensive care, maternity), patient location (e.g. pediatrics, ER,maternity), and/or time stamps such as sample collection, sampleordering, time received at central receiving, central receiving sort,transport to lab and/or accession of sample. These time stamps providereal-time monitoring by the instrument software of pending test orderturn-around time.

LIS Interchange→Clinical Instrument's CPU

The automated nucleic acid testing system with communicationcapabilities is referred to as a “Clinical instrument” ofsample-to-answer system. After the LIS interchange receives the testorder, it sends it to the sample-to-answer system's (clinicalinstrument's) CPU in the base station.

The sample-to-answer system supports both serial and ethernet/RJ45input/output connections to one or more hospital LIS.

The Sample Stability feature or Sample Stability time allows the user tospecify the stability time on a per assay basis. The software tracks PTOorders and sends an alert notification when an order has violated thethreshold for sample stability. The sample-to-answer system includes“cleanup rules” to automatically delete outstanding pending test orderse.g. delete a PTO if it is in the Pending Test Order queue for apredetermined time (called max PTO time), preferably for more than oneweek, preferably for more than two weeks.

These bi-directional LIS capabilities improve PTO to detection reportturnaround time, reduce labor costs, and eliminate potentialtranscription errors.

The communication from the LIS interchange to the detection device's CPUcan be referred to as the clinical instrument test order.

Reporting Results

Detection Reports

After the sample is run in a detection system, a result is generated. Aresult is generated if the amplified target DNA/neucleic acid hybridizesto its complementary signal probe and capture probes. The CPU in thebase station then sends (either automatically or manually) a detectionreport (also referred to as a result report or test results) to the LISinterchange which converts the detection report into a physician testresult report and sends the physician test result report to thehospital's LIS which then sends the physician result report to thephysician or directly to the physician. The detection report/physiciantest result sent to the hospital's LIS or to the physician can includedetected targets, non-detected targets, invalid results and/or controldata. The sample-to-answer system can either auto release allinformation or hold all information for manual release. Alternatively,the sample-to-answer system can auto release some detection reports andhold some detection reports for manual release. For example, detectedand non-detected targets can be auto-released while invalids can bemanually released (i.e., released only after a lab supervisor approvesfor release). If the detection report shows 3 (triple infection) orfewer targets were identified/detected the detection report willautomatically release to the hospital's LIS/physician. If the detectionreport shows greater than 3 (i.e. 4 or more) targets wereidentified/detected the report will be flagged, a multiple infectionerror alert (also called an alert notification) can be sent to theoperator or physician and the sample can be automatically re-run. Thedetection report includes the assay ordered. If a cartridge is insertedthat does not match the assay ordered (e.g. a gram-negative assay isordered but a respiratory assay is inserted) a “mismatch alert” is sentto the operator and/or physician and/or the additional target is notedin the detection report. Anomalous results that are not auto-releasedcan require a manager signature before manual release. Such reportingminimizes the risk of reporting errors.

The detection report can include time stamps such as sample collectiontime, sample ordering time, transport to central receiving time, centralreceiving sort time, transport to lab time, accession of sample time,time to process, and time to detection. FIG. 3 includes an“order-to-report” timeline. The clock time stamps are commonlydocumented in hospital and laboratory information systems.

The automated result reporting (at order entry and results reporting)eliminates transcription errors and ensures actionable results arereturned to physicians as soon as possible. Sample results are reportedin about 60-90 minutes after the start of running the sample, this isreferred to as time to result (See FIG. 2) or sample to result.Preferably, sample results are reported in about 60 minutes after thestart of running the sample. Preferably, sample results are reported inunder 90 minutes after the start of running the sample. Preferably,sample results are reported upon test completion. A detection report issent immediately after the pathogen is identified by the detectionsystem.

The sample-to-answer system allows the operator to include comments inthe detection report called detection report comments, e.g., to specifyif the assay ordered matched the target detected, if the assay ordereddoes not match the target detected, if an additional target was detectedin addition to the target for the assay ordered, if a second assay isrecommended, if a resistance gene was identified, suggest a course oftreatment such as antibiotic.

Control Reports

Control reports or Control summary reports are generated based on theassay, test frequency and lot of cartridges from the supplier. Controlreports provide information about the number of samples run, and whencontrol runs are needed. When a control run is processed, the reportshows the expected and actual result, if the control passed or failed.Control runs are typically run every 30 days or every lot change. Thesample-to-answer system alerts to the operator 48 and/or 24 hours beforea control run is needed.

System Usage Report

The system usage report provides analytics around system usage data andperformance based on a specified date range. For example, the systemusage report will show if higher or lower than average samples were run,if higher or lower than expected samples were run, if a bay has not beenutilized, etc. System Usage Reports can be printed from the ClinicalInstrument or remotely by the clinical instrument's provider.

Service Notification Report

A service notification report is a report sent to the clinicalinstrument's provider to request remote access to the clinicalinstrument to trouble shoot errors such as when a device has exceededdowntime for a month, exceeded invalid runs, mean time to failure is toohigh, no LIS connectivity etc.

Alerts

The sample-to-answer system includes a number of automatic alerts.

A Remote Practitioner Alert is an alert sent to practitioners to notifythem that test results are available.

A Non-Operator Alert is an alert sent to non-operators such aslab-managers, directors of labs etc. regarding test results.

A Reportable Organism Alert is an alert sent based on a user-definedreportable organisms. For example, if a patient is diagnosed with aninfectious disease, then an alert can be sent to the Department ofHealth.

A Turnaround Time Violation Alert is an alert sent to the physician,operator or lab manager when the predetermined turnaround time isviolated.

A Sample Stability Time Violation Alert is an alert sent to thephysician, operator or lab manager that the sample stability time wasviolated.

A Duplicate Accession ID Alert is an alert notifying the operator that asample with the same accession number was already run. Since each sampleshould have its own accession number, the operator should review for apossible error.

A Multiple Infection Error Alert is an alert to notify the operator thatthere are 4 or more co-infections detected and the sample should bere-run.

A Mismatch Alert is an alert sent to the operator or physician that atarget is detected which does not match the assay ordered (e.g. agram-negative assay is ordered but a fungal infection is identified).The mismatch can be the only target detected or can be in addition to atarget expected to be detected by the assay ordered. When a mismatchalert is sent the sample can be automatically re-run on the assayordered or on another assay which matches the mismatch. For example, ifthe assay ordered was a BCID-GP assay but a fungal target wasidentified, the BCID-GP assay can be re-run and/or a BCID-FP assay isrun.

User Interface

The detection system includes a user interface comprising a touch screendisplay having a plurality of bay icons, each icon uniquelycorresponding to one of said plurality of bays. The user interfacefurther includes hour, minute and second countdown timer on the bay iconto show the time left until a result will be reported.

Additionally, the user interface will display the bay status (whetherthe bay is empty, the presence or absence of a cartridge, whether thecartridge assay is underway, assay complete, and a process error) evenwhile the user is logged out.

The user interface audible clicks by default on a virtual keyboard.

The user interface allows batch printing of reports.

QC Results

Monitoring and reporting quality control is both a requirement and abest practice to ensure the accuracy of patient testing results andcompliance with lab standards. With on-board QC tracking capabilities,the sample-to-answer system provides safeguards to ensure labs not onlyrun controls when required but can easily track and report compliance.Indeed, the base station itself retains onboard QC test records to helpensure the lab runs controls when required. As discussed above, controlreports are sent if an external control is due in 48 hours and/or 24hours.

The sample-to-answer system can prevent new runs if the detection systemhas not been qualified. This means that if a new lot is provided and acontrol should be run on the clinical instrument before running apatient sample, the instrument will prevent a patient sample test untilthe control is run.

The Sample-to-answer system further supports the release of QC resultsto the hospital LIS either automatically or manually.

Further, patient data is automatically removed in all exported run data(troubleshooting logs and raw data calculations such as nA signal fromtargets, non-detected targets, controls etc) for HIPPA compliance.

The sample-to-answer system tracks and reports required preventativemaintenance. Such systems maximize lab efficiency by reducingadministrative overhead.

Compliance and Data Management

The sample-to-answer system provides the following compliance and datamanagement tools: Integrated data analytics to easily monitor labperformance, on-demand epidemiology reports for export and simplifiedanalysis in Excel (including disease prevalence in a geographic area);and fully configurable, auto-release of test results (detected targetsas well as non-detected targets). All of these unique capabilities ofthe sample-to-answer system allow Lab Directors to reduce their timespent on routine administrative tasks and focus their limited resourceson high-value activities that impact patient care and the bottom line.

Specifically, on demand Epidemiology reports can be run from each basestation individually or collectively from all of the base stations runin the laboratory via the LIS.

Remote Service Capability

The sample-to-answer system includes remote service capability tominimize system downtime and ensure patients and physicians have accessto rapid test results. Remote service may be needed when the clinicalinstrument has exceeded downtime for a month, exceeded invalid runs,mean time to failure is too high, no LIS connectivity etc.

Positive Patient ID

The sample-to-answer system's positive patient ID feature reduces thepotential for patient sample mix-up. Positive patient ID is described inmore detail in U.S. Pat. No. 9,500,663 which is herein incorporated inits entirety by reference. Specifically, two machine-readableinformation tags (or patient identification tags) are arranged on thecartridge and encoded with cartridge-identifying information, where theinformation encoded in the second tag corresponds to the informationencoded in the first tag and is read by a device within the sampleprocessing instrument.

After a first machine-readable information tag on the outside of thecartridge is scanned, the cartridge can be loaded into any bay at anytime, this is referred to as “random access” or “random and continuousbay access” or “unassigned” or un-delegated” or un-allocated” orunspecified” and the like. Stated another way, the cartridge need not beloaded into a specified bay. In this way, loading errors are avoided.Once the cartridge is loaded, the bay's CPU reads a secondmachine-readable information tag and confirms it matches the firstmachine-readable information tag.

With the sample-to-answer system, labs and physicians can haveconfidence that they have the right patient, with the right test, andthe right result every time.

The sample-to-answer system can be further understood by the followingnumbered paragraphs:

Paragraph 1. An in vitro method for reporting test results to a hospitalLIS comprising: obtaining a test order from a hospital's laboratoryinformation system (LIS); conveying the test order to a sample-to-answersystem; receiving and processing a sample from the hospital associatedwith the test order; generating a detection report identifying 1 or 2 or3 human pathogens in the sample; and automatically sending the detectionreport to the hospital LIS.

Paragraph 2. An in vitro method for reporting test results to a hospitalLIS comprising: obtaining a physician test order; generating a pendingtest order (PTO); conveying the PTO to a hospital's laboratoryinformation system (LIS); generating a test order; conveying the testorder to an LIS interchange; generating a clinical instrument testorder; conveying the clinical instrument test order to a detectiondevice; receiving and processing a sample from the hospital associatedwith the physician test order; generating a detection report identifying1 or 2 or 3 human pathogens in the sample; automatically sending thedetection report to the LIS interchange; converting the detection reportto a physician test result; automatically sending the physician testresult to the hospital LIS.

Paragraph 2: The method of any preceding paragraph, wherein thedetection report is automatically sent to the hospital within 90 minutesof when the sample processing began.

Paragraph 3: The method of any preceding paragraph, wherein thedetection report includes one or more time stamps selected from thegroup comprising time stamps such as sample collection, sample ordering,transport to central receiving, central receiving sort, transport tolab, accession of sample, time to process, or time to detection report.

Paragraph 4. The method of any preceding Paragraph, wherein when thedetection report identifies four or more pathogens, a multiple infectionerror alert is sent to the physician.

Paragraph 5. The method of any preceding Paragraph, wherein an alert issent to practitioners that the detection report is available.

Paragraph 6. The method of any preceding Paragraph, wherein anepidemiology report is generated by the clinical instrument.

Paragraph 7. The method of any preceding Paragraph, wherein the sampleis associated with a patient identification tag such as anelectronically-readable tag, a wirelessly-readable tag, a radiofrequency identification (RFID) tag or an electrically EPROM (EEPROM)tag.

Paragraph 8. An in vitro method for reporting the detection of a humanpathogen and/or genetic material thereof comprising: obtaining a sample;loading the sample in a detection system; subjecting a sample to asingle multiplex polymerase chain reaction (PCR) thereby detecting ahuman pathogen; generating a report containing the identification of thepathogen; automatically delivering the report from the detection systemto an LIS interchange; delivering the report from the LIS interchange toa hospital LIS.

EXAMPLES

The invention is demonstrated in practical embodiments by the followingexamples. The embodiments disclosed therein relate to potentiallypreferred embodiments of the invention and are not intended to limit thescope of the invention.

Example 1: Gram-Positive Panel, Blood Culture Contamination

False positive Enterococcus faecalis, Pan-GN and Pan-candida signalswere observed when Applicants ran the sLRM GP assay. Applicantsinvestigated whether the blood culture matrix was the source of thecontamination.

Desired blood culture bottles were collected. The rubber sealer of eachblood culture bottle was cleaned with ethanol before puncturing it witha needle. 75 uL from each bottle was aspirated. sLRM was performed (Beadbeater sample- to lyse cell, add 300 uL lysis buffer, 500 uL bindingbuffer-wait 2 min, and wash with 150 uL wash buffer). 100% of the washedmagnetic beads were loaded onto the cartridge. H1 and H3 (annealingheaters) were run at 61.5° C.

A preliminary test of NTC sLRMs (bottle matrix with no blood orbacterial targets) showed high false positive signals for Enterococcusfaecalis, Pan-candida, and Pan-GN but buffer alone runs did not (seeFIG. 4). As a result it was determined that contamination is coming fromthe bottle matrix.

To follow up, 13 negative (no blood or bacterial targets) blood matriceswere screened for contaminants. Desired blood culture bottles werecollected. 1000 uL of Bottle matrix was collected and the sample beadbeated. 1 S. pombe lyo pellet (5e5 Colony Forming Unit (CFU)/bead)(control), 75 uL of Bead beaten sample and 300 uL Lysis Buffer was addedto an Eppendorf tube. N=8 per bottle type. Waited 5 minutes then added500 uL of Binding buffer, rotate tubes. Centrifuge briefly and put thetubes onto magnetic racks. Waited for 1-2 minutes and aspirated theliquid using 1000 uL pipettes. Wash the beads with 150 uL of washbuffer, remove residual wash buffer after briefly centrifuging thetubes. Resuspended beads with 150 uL of wash buffer. Loaded 100% ofbeads onto open bay runs or store them @ 4 C. Table 4 below summarizesthe Pan-GN and Pan-candida contaminates identified in the negative bloodmatrices.

TABLE 4 Blood Culture Bottle Contaminants Blood Culture Bottle Pan-GPand Pan-candida Brand Types Contaminants identified 1 BACTEC PlusAnaerobic/F Pan-candida 2 BACTEC Standard/10 Aerobic/F Pan-candida 3BACTEC Standard Anaerobic/F Pan-candida 4 BACTEC Plus Aerobic/FEnterococcus Faecalis, Enterococcus spp. Pan candida 5 BACTEC PediatricPlus Pan-candida 6 BACTEC Lytic/10 Anaerobic/F Enterococcus Faecalis-Pan-candida 7 BacT/ALERT SA Standard Aerobic None 8 BacT/ALERT SNStandard Anaerobic Pan-Candida, Staphylococcus 9 BacT/ALERT FA PlusPan-candida 10 VersaTREK REDOX 1 Aerobic Pan-candida 11 VersaTREK REDOX2 Anaerobic Pan-candida 12 BacT/ALERT FN Plus Pan-candida 13 BacT/ALERTPF Plus Pan-candida

Blood culture matrices that were known to have contaminants were thenevaluated on the BCID-GP cartridge. FIG. 5a shows Enterococcus faecalis,FIG. 5b shows Staphylococcus, FIG. 5c shows Enterococcus (genus) andFIG. 5d shows Pan-Candida were detected when negative blood culturematrices (no blood or bacterial targets) were tested on a BCID-GPcartridge.

Blood culture bottles were sent out for DNA testing by sequencing toconfirm DNA was coming from the bottle and not another source. Table 5below details the percentage of DNA in the empty bottle attributable tothe contaminate.

TABLE 5 Percentage of DNA In The Empty Bottle Attributable To TheContaminate BD BD Genus Anerobic Aerobic Bacillus 17.838% 16.61% Streptococcus 4.838% 5.51% Enterococcus 0.568% 0.90% Micrococcus 0.199%0.74% Listeria 0.521% 0.73% Acinetobacter 0.337% 0.62% Proteus 0.655%0.78% Stenotrophomonas 0.024% 0.30% Propionibacterium 0.046% 0.27%Morganella 0.124% 0.24% Staphylococcus 0.170% 0.18% Corynebacterium0.114% 0.17% Serratia 0.062% 0.14% Pseudomonas 0.082% 0.13% Pantoea0.052% 0.11% Lactobacillus 0.020% 0.08% Bacteroides 0.006% 0.07%Cronobacter 0.026% 0.04% Citrobacter 0.001% 0.00% Klebsiella 0.002%0.00% Salmonella 0.001% 0.00% Enterobacter 0.000% 0.0004%  Neisseria0.000% 0.0004%  Prevotella 0.019% 0.00%

Example 2: Gram-Positive Enterococcus faecalis DNA Contamination

In order to eliminate Enterococcus faecalis signals coming from theblood culture matrices, reduced PCR cycling was evaluated. Two strainsof Enterococcus faecalis (Enterococcus faecalis ATCC19433 andEnterococcus faecalis ATCC49532) at 1× Limit of Detection (LoD) (1×105CFU/mL) were PCR cycled 40 and 35 times. Specifically, 10 sLRM were madefor each strain type and dilute to 50% beads. FIG. 6A shows thatEnterococcus faecalis contamination signals are reduced but noteliminated with 35 cycles and the S. pombe internal control is stilldetected (See FIG. 6B).

Next, 30-cycle PCR was evaluated. FIGS. 7a and 7b show 2 types ofnegative blood culture bottles (Bactec plus aerobic and Peds plus/f),using 75 uL direct input tested on open bay runs. False positive signalfrom blood culture bottles is eliminated using a 30-cycle PCR. Only thepositive internal controls (S. pombe and DT9) were detected. FIGS. 7cand 7d show that detection is possible (although weak) at 1×LOD (1×105CFU/mL) Enterococcus faecalis run on sLRMs using a 30-cycle PCR.

Because Enterococcus faecalis will run with a 30-cycle PCR, it wascombined/pooled with the Pan-GN primers which also cycle 30 times.

Example 3: Gram-Positive, Streptococcus Spp. and P. acnes Contamination

It was observed that the BCID-GP panel also detects common gram-positiveorganisms or nucleic acid found in the blood culture bottles. Detectionof these organisms from the environment leads to false positives.Streptococcus spp. and P. acnes lead to the most number of falsepositives. To mitigate the risk of false positives, Streptococcus and P.acnes were amplified with decreased cycling (30 or 35 cycles from 40)Amplification conditions are as follows:

TABLE 6 Bench PCR conditions for primer optimization PCR ComponentWorking Concentrations PCR Buffer 1X + MgCl2 1.25X and 3 mM,respectively dNTPs 0.8 mM AptaTaq LDX 4U/rxn Enhancer with 0.1% 1X TweenMultiplex Primer Mix 1X Total PCR Reaction 2 μL

TABLE 7 PCR Cycling conditions Cycles Temp. Time Stage 1  1X 95° C. 20″Stage 2 40X 95° C.  3″ 35X 60° C. 18″ 30X

Tests using a BDIC-GP cartridge spotted with P. acnes primers show thatP. acnes false positives are detected with 40 and 35 cycles buteliminated with 30 cycle PCR (FIG. 8). Thus, decreasing cycleseliminates contamination detection of P. acnes coming from negativeblood culture matrices.

Next, Applicants analyzed whether 30 cycles allows sufficientamplification to detect the targets at 1×LOD. Negative blood culturematrixes (10 mL of blood in a blood culture bottle) were spiked with P.acnes. The P. acnes was then bead beaten. 100 uL of bead beaten sampleand 1 S. pombe lyo pellet was then added to 300 uL of lysis buffer andincubated for 1 minute. Following that 500 uL of binding buffer wasadded and incubated for 2 minutes. The magnetic beads were thencollected and washed once with 150 uL of wash, they were thenresuspended in 150 uL of wash and transferred to open bays.

FIG. 9 shows that when 30 PCR cycles are used, the sLRM assay is stillcapable of detecting P. acnes at 1×LOD (1×106 CFU/mL). (FIGS. 9a and 9b).

Six representative Streptococcus species were also tested and shown notto produce false positive signals using 30 PCR cycles on BCID-GPcartridges (data not shown) yet capable of 1×LOD (1×106 CFU/mL)detection. FIG. 10.

Primer optimization: Because Streptococcus spp. and P. acnes havereduced PCR cycling to avoid false positives while maintaining targetsensitivity, they were placed in their own primer pool on the PCB board.The primers were combined with Internal Control 1 (IC1) template and IC1primers. IC1 is a synthetic ssDNA sequence with zero mismatches in theprimer binding regions. Streptococcus spp. and P. acnes primers wereinitially evaluated at their original working concentrations of 250 nMbut were increased to 500 nM for improved performance. Three P. acnesstrains and two Streptococcus species were tested on open bays. Alltargets were detected at 1×LOD (1×106 CFU/mL) (FIG. 11). While the P.acnes signal is less robust than the Streptococcus spp. signal, it isabove the signal threshold (10 namps).

TABLE 8 Multiplex Pool formulation Working Organisms and Rev [Primer]Amplicon Pool Drug Resistance Target For Primer ID Primer ID nM size bpMP5 propionibacterium rpoB- D12933 D12936 500 183 Prop Streptococcus 16sD12193 D12945 500 110 spp. RNA- Strep IC1 IC1 D19507- D19506- 250 99 H3H3 Internal Control 1 D19505 1000 copies

In order to finalize the multiplex primer pool, it was necessary toconfirm whether contamination signals were eliminated. NTC sLRMs wererun with 100% bead loading to evaluate contamination levels. The resultsdemonstrated that no Streptococcus spp or P. acnes signals were detectedwhile the S. pombe and IC1 control signals were detected. FIG. 12.

Example 4: Gram-Positive, Contamination Mitigation

Next Applicants evaluated 37-cycle PCR for all targets to reduce oreliminate contamination from blood matrix bottles. Three types ofbottles were tested (Bactec Pediatric Plus/F, Bactec Aerobic Plus/F,Bactec Anaerobic Lytic/10) with and without blood.

Surprisingly, when PCR cycles are reduced from 40 to 37, most bloodmatrix contamination is eliminated. FIG. 13.

Example 5: Gram-Positive, Detuning to Eliminate Blood CultureContamination

In light of the above experiments, Applicants reduced all cycling to 35or 30 cycles. Even with the reduction in cycles, false positives werestill detected. For example, Corynebacterium was reduced from 40 to 35and then to 30 cycles but false positives persisted. Applicants thendropped the primer concentration by 50% to 250 nM and the falsepositives were eliminated. Enterococccus false positives were eliminatedwhen PCR cycles were dropped from 40 to 35 cycles and primerconcentration was reduced by 50% to 250 nM. S. anginosus false positiveswere eliminated when PCR cycles were dropped from 40 to 35 cycles andprimer concertation was reduced by 75% to 250 nM. The primerconcentration for the other targets ranges from 125 to 1000 nM. Assummarized in the table below, Applicants were surprisingly able to maketheir BCID-GP assay less sensitive, to eliminate or reduce detection ofcontaminants in the sample by “detuning” which in some cases involvedonly the reduction in the number of cycles and in other cases involvedthe combined reduction in cycling and reduction in primer concentrationand thresholding.

TABLE 9 Assay thresholds Target Before detuning After detuning (nA) Bcereus not detected not detected 20 B subtilis detected not detected 20Corynebacterium detected not detected 10 Enterococccus detected notdetected 20 E faecalis detected not detected 20 E faecium detected notdetected 20 and requires Entero call Lactobacillus not detected notdetected 20 Listeria not detected not detected 20 L monocytogenes notdetected not detected 20 Micrococcus detected not detected 30 P acnesdetected not detected 50 Staphylococcus detected not detected 50 Saureus detected not detected 20 S epidermidis detected not detected 20 Slugdunensis not detected not detected 20 Streptococcus detected notdetected 70 S agalactiae detected not detected 100 S anginosus detectednot detected 20 S pneumoniae detected not detected 20 S pyogenesdetected not detected 50 S maltophilia detected not detected 30 Pan-GNdetected not detected 100 Pan-Candida not detected not detected 10 mecAdetected not detected 20 mecC not detected not detected 20 vanA notdetected not detected 20 vanB not detected not detected 20

Example 6, Gram-Positive, Use of Benzonase to Remove Bottle CultureContaminates

In the above Examples, the goal was to amplify PCR products in a singlePCR run under conditions appropriate for the substantial reduction orelimination of electrochemical detection of contaminating pathogenand/or genetic material present in the sample. Next Applicants sought toreduce or eliminate the concentration of contaminating nucleic acids inthe sample. The invention employs nucleases to remove contaminatingnucleic acids. Exemplary nucleases include BENZONASE®, PULMOZYME®; orany other DNase or RNase commonly used within the art.

Enzymes such as BENZONASE® degrade nucleic acid and have no proteolyticactivity. As with all endonucleases, BENZONASE® hydrolyzes internalphosphodiester bonds between specific nucleotides. Upon completedigestion, all free nucleic acids present in solution are reduced tooligonucleotides 2 to 4 bases in length. It is well known that nucleicacids may adhere to cell derived particles such as viruses. BENZONASE®has been used to purify samples with viral targets. See e.g. U.S. Pat.No. 9,428,768 which is herein incorporated in its entirety by reference.What is not known is whether BENZONASE® would be useful in removingnon-viable organisms (bacteria or fungi) found in growth media withoutinterfering with detection of a bacterial or fungal target in thesample.

15 clinical samples were tested on the BCID-GP Panel that had previouslygiven Enterococcus faecium false positives. Specifically, samples weretreated with Benzonase+1 mM MgCl2 for 5 min. at 37 C or roomtemperature. Benzonase eliminated all false positives. Room temperatureincubation was as effective as 37 C incubation

Next, the blood bottle, BacT/Alert FA Plus blood matrix, which hasBacillus subtilis DNA contamination, was treated with Benzonase asdescribed above. Without Benzonase treatment, the blood culture matrixgave background signals of 1.9-10.9 nA but 9 replicates ofBenzonase-treated matrix did not give any signal greater than 0.2 nA,thus eliminating false positive signals.

Next, whether Benzonase impacts target detection was tested. 5 clinicalsamples were tested on the BCID-GN panel that had previously given falsepositives. Samples were treated with Benzonase at room temperature for 5min and 2 hr. All false positives signals were eliminated and all truetargets were detected with 5 and 2 hr treatment, but several targetsignals decreased with the 2 hr. treatment.

Next, 3×LOD (3×106 CFU/mL) Multiplex Primer Pool (MP) mix #3 (comprisingEscherichia coli, Lactobacillus paracasei, Streptococcus pyogenes,Listeria innocua, Candida albicans) and blood/BC matrix with 0, 5′ or 2hrs Benzonase treatment was tested and run on BCID-GP cartridge with 35,30 and 40 PCR cycles. All LOD targets were detected with 5′ and 2 hrbenzonase treatment with both 30 and 35 PCR cycling parameters. Not allcontamination is eliminated with 40-cycle PCR.

BENZONASE® is well suited for reducing nucleic acid and/or non-viablebacterial and fungal organisms without adversely impacting the detectionof clinically relevant bacteria (gram-positive/gram-negative) or fungaltargets.

Example 7: Gram-Positive Panel, Limit of Detection (AnalyticalSensitivity)

The BCID-GP Multiplex primer pool and PCR cycles are shown in FIG. 14.Each of the 8 PCR drops contains an internal control S. pombe is thecontrol target in PCR drops 1-4 (30-cycle PCR) Synthetic Control 1 (SC1)is the control target in PCR drops 5-8 (35-cycle PCR).

The PCR cycling conditions are as follows:

TABLE 10 BCID-GP PCR cycling Denature 95.5° C. Anneal/Extend 65.0° C.Cycle No. Hot Start 30 sec. Step 1  3 sec. 30 sec. 1-30 or 1-35

This primer pool and PCR cycling are used for Examples 7-10. The BCID-GPcartridge layout is shown in FIG. 15 and was also used in Examples 7-10.

The limit of detection (LoD), or analytical sensitivity, was identifiedand verified for each assay on the BCID-GP Panel using quantifiedreference strains. To facilitate testing, five organism mixes were madeat varying concentrations and serial dilutions were prepared insimulated blood culture sample matrix which is defined as the matrixfrom a negative blood culture bottle mixed with whole blood and EDTA inthe same ratio as the manufacturer recommends for blood culture. Atleast 20 replicates per target were tested for each condition. The limitof detection was defined as the lowest concentration of each target thatis detected in >95% of tested replicates. The confirmed LoD for eachBCID-GP Panel organism is shown in Table 11.

TABLE 11 LoD Results Summary LoD Target Organism Strain ConcentrationBacillus cereus Group Bacillus cereus ATCC 21769 1 × 10⁵ CFU/mL Bacillussubtilis Group Bacillus subtilis ATCC 55614 1 × 10⁵ CFU/mLCorynebacterium Corynebacterium ATCC 43735 1 × 10⁶ CFU/mL striatumEnterococcus Enterococcus ATCC 25788 1 × 10⁵ CFU/mL casseliflavusEnterococcus faecalis Enterococcus faecalis ATCC 51575 1 × 10⁶ CFU/mL(vanB+) Enterococcus faecium Enterococcus faecium ATCC BAA- 1 × 10⁶CFU/mL (vanA+) 2317 Lactobacillus Lactobacillus paracasei ATCC 25598 1 ×10⁵ CFU/mL Listeria Listeria monocytogenes ATCC 10890 1 × 10⁵ CFU/mLListeria monocytogenes Listeria monocytogenes ATCC 10890 1 × 10⁵ CFU/mLMicrococcus Micrococcus luteus ATCC 19212 1 × 10⁶ CFU/mLPropionibacterium Propionibacterium ATCC 6919 1 × 10⁸ CFU/mL acnes acnesStaphylococcus Staphylococcus NRS 879 1 × 10⁵ CFU/mL lugdunensisStaphylococcus aureus Staphylococcus aureus ATCC BAA- 1 × 10⁵ CFU/mL(mecC+) 2313 Staphylococcus Staphylococcus ATCC 35983 1 × 10⁵ CFU/mLepidermidis epidermidis (mecA+) Staphylococcus Staphylococcus NRS 879 1× 10⁵ CFU/mL lugdunensis lugdunensis Streptococcus Streptococcus ATCCBAA-475 1 × 10⁵ CFU/mL pneumoniae Streptococcus Streptococcus ATCC 124011 × 10⁶ CFU/mL agalactiae agalactiae Streptococcus Streptococcusanginosus ATCC 9895 1 × 10⁵ CFU/mL anginosus group StreptococcusStreptococcus ATCC BAA-475 1 × 10⁵ CFU/mL pneumoniae pneumoniaeStreptococcus pyogenes Streptococcus pyogenes ATCC 12384 1 × 10⁵ CFU/mLmecA Staphylococcus ATCC 35983 1 × 10⁴ CFU/mL epidermidis (mecA+) mecCStaphylococcus aureus ATCC BAA- 1 × 10⁴ CFU/mL (mecC+) 2313 vanAEnterococcus faecium ATCC BAA- 1 × 10⁴ CFU/mL (vanA+) 2317 vanBEnterococcus faecalis ATCC 51575 1 × 10⁴ CFU/mL (vanB+) Pan CandidaCandida albicans ATCC 24433 1 × 10⁶ CFU/mL Candida glabrata ATCC 66032 1× 10⁶ CFU/mL Pan Gram-Negative Escherichia coli ATCC 4157 1 × 10⁶ CFU/mLStenotrophomonas ATCC 13636 1 × 10⁶ CFU/mL maltophilia

Example 8, Gram-Positive, Analytical Reactivity (Inclusivity andExclusivity)

A panel of 158 strains/isolates representing the genetic, temporal andgeographic diversity of each target on the BCID-GP Panel was evaluatedto demonstrate analytical reactivity. Each strain was tested intriplicate at 1×108 CFU/mL while each fungus was tested at 1×106 CFU/mLin simulated sample matrix.

All of the 158 strains/isolates tested for inclusivity were detected bythe BCID-GP Panel. Results of analytical reactivity are shown in Table12.

Analytical Specificity (Cross-Reactivity and Exclusivity)

Cross-reactivity of on-panel analytes was evaluated using data generatedfrom the Analytical Reactivity study. Cross-reactivity of off-panelorganisms was evaluated by testing a 30 member panel, containingclinically-relevant bacteria and fungi. Bacterial targets were tested ata concentration of >1×109 CFU/mL while fungi were tested at aconcentration of >1×107 CFU/mL. In three cases where >1×109 CFU/mL couldnot be achieved in culture for bacteria, a two-fold dilution of thestock material was used as reflected in Table 12. Table 12 summarizesthe results of the on-panel organism strains tested. Each on-panelstrain was tested in triplicate. Table 13 summarizes the results of theoff-panel fungal and bacterial strains tested. No cross-reactivity wasobserved for any of the off nor on-panel organisms with any of theassays.

TABLE 12 Analytical Reactivity (Inclusivity, Cross-Reactivity, andExclusivity) Results Percent Percent Cross-Reactivity Organism StrainDetected Positivity Result Bacillus cereus ATCC 10876 100% 100% NotObserved Bacillus thuringiensis ATCC 10792 100% 100% Not Observed ATCC35646 100% 100% Not Observed Bacillus ATCC 23350 100% 100% Not Observedamyloliquefaciens ATCC 23845 100% 100% Not Observed Bacillus atrophaeusATCC 6455 100% 100% Not Observed ATCC 6537 100% 100% Not ObservedBacillus licheniformis ATCC 21039 100% 100% Not Observed ATCC 21667 100%100% Not Observed Bacillus subtilis ATCC 15561 100% 100% Not ObservedCorynebacterium ATCC 39255 100% 100% Not Observed diphtheriae ATCC 53281100% 100% Not Observed Corynebacterium ATCC 51799 100% 100% Not Observedulcerans Corynebacterium ATCC BAA- 100% 100% Not Observed jeikeium 949ATCC BAA- 100% 100% Not Observed 950 ATCC 43734 100% 100% Not ObservedCorynebacterium ATCC 43044 100% 100% Not Observed urealyticumCorynebacterium ATCC 7094 100% 100% Not Observed striatum Enterococcusavium ATCC 14025 100% 100% Not Observed Enterococcus gallinarum ATCC49608 100% 100% Not Observed Enterococcus hirae ATCC 49479 100% 100% NotObserved Enterococcus ATCC 100% 100% Not Observed casseliflavus 700327Enterococcus raffinosus ATCC 49464 100% 100% Not Observed EnterococcusATCC 43076 100% 100% Not Observed saccharolyticus Enterococcus faecalisATCC 14506 100% 100% Not Observed ATCC 19433 100% 100% Not Observed ATCC29200 100% 100% Not Observed ATCC 49149 100% 100% Not Observed ATCC49332 100% 100% Not Observed ATCC 49452 100% 100% Not Observed ATCC49474 100% 100% Not Observed ATCC 49532 100% 100% Not ObservedEnterococcus faecalis ATCC BAA- 100% 100% Not Observed (vanB+) 2365Enterococcus faecium ATCC 19953 100% 100% Not Observed ATCC 23828 100%100% Not Observed ATCC 27270 100% 100% Not Observed ATCC 27273 100% 100%Not Observed ATCC 35667 100% 100% Not Observed ATCC 49224 100% 100% NotObserved ATCC 49624 100% 100% Not Observed Enterococcus faecium ATCC51559 100% 100% Not Observed (vanA+) ATCC 100% 100% Not Observed 700221ATCC BAA- 100% 100% Not Observed 2316 ATCC BAA- 100% 100% Not Observed2318 ATCC BAA- 100% 100% Not Observed 2319 ATCC BAA- 100% 100% NotObserved 2320 Enterococcus faecium ATCC 51858 100% 100% Not Observed(vanB+) Listeria monocytogenes ATCC 13932 100% 100% Not Observed ATCC19111 100% 100% Not Observed ATCC 19112 100% 100% Not Observed Listeriainnocua NCTC 11288 100% 100% Not Observed Listeria ivanovii ATCC 19119100% 100% Not Observed ATCC BAA- 100% 100% Not Observed 139 Listeriaseeligeri ATCC 35967 100% 100% Not Observed Listeria welshimeri ATCC35897 100% 100% Not Observed Lactobacillus casei ATCC 334 100% 100% NotObserved ATCC 39392 100% 100% Not Observed Lactobacillus paracasei ATCC27092 100% 100% Not Observed Lactobacillus rhamnosus ATCC 39595 100%100% Not Observed ATCC 53103 100% 100% Not Observed Micrococcus luteusATCC 400 100% 100% Not Observed ATCC 4698 100% 100% Not ObservedMicrococcus ATCC 7468 100% 100% Not Observed yunnanensisPropionibacterium acnes ATCC 11827 100% 100% Not Observed StaphylococcusATCC 100% 100% Not Observed gallinarum 700401 Staphylococcus ATCC 29970100% 100% Not Observed haemolyticus ATCC 31874 100% 100% Not ObservedStaphylococcus hominis ATCC 27844 100% 100% Not Observed ATCC 100% 100%Not Observed 700236 NRS 871 100% 100% Not Observed Staphylococcus hyicusATCC 11249 100% 100% Not Observed Staphylococcus lentus ATCC 100% 100%Not Observed 700403 Staphylococcus capitis ATCC 35661 100% 100% NotObserved NRS 866 100% 100% Not Observed Staphylococcus ATCC 43764 100%100% Not Observed chromogenes Staphylococcus cohnii ATCC 29974 100% 100%Not Observed Staphylococcus vitulinus ATCC 51161 100% 100% Not ObservedStaphylococcus pasteuri ATCC 51129 100% 100% Not Observed Staphylococcussimulans ATCC 27850 100% 100% Not Observed ATCC 27851 100% 100% NotObserved Staphylococcus aureus ATCC 11632 100% 100% Not Observed ATCC14775 100% 100% Not Observed ATCC 29213 100% 100% Not Observed ATCC29247 100% 100% Not Observed ATCC 6538P 100% 100% Not Observed ATCC25923 100% 100% Not Observed Staphylococcus aureus NRS 383 100% 100% NotObserved (mecA+) NRS 384 100% 100% Not Observed NRS 385 100% 100% NotObserved NRS 387 100% 100% Not Observed NRS 483 100% 100% Not ObservedNRS 484 100% 100% Not Observed NRS 643 100% 100% Not Observed NRS 645100% 100% Not Observed NRS 653 100% 100% Not Observed ATCC 100% 100% NotObserved 700698 ATCC 100% 100% Not Observed 700699 ATCC BAA- 100% 100%Not Observed 1707 ATCC BAA- 100% 100% Not Observed 40 ATCC BAA- 100%100% Not Observed 42 ATCC BAA- 100% 100% Not Observed 43 NRS 382 100%100% Not Observed NRS 386 100% 100% Not Observed NRS 647 100% 100% NotObserved NRS 654 100% 100% Not Observed NRS 655 100% 100% Not ObservedNRS 657 100% 100% Not Observed NRS 659 100% 100% Not Observed NRS 648100% 100% Not Observed NRS 651 100% 100% Not Observed StaphylococcusATCC 49134 100% 100% Not Observed epidermidis ATCC 100% 100% NotObserved 700583 NCIMB 8853 100% 100% Not Observed Staphylococcus ATCC49461 100% 100% Not Observed epidermidis (mecA+) Staphylococcus ATCC49576 100% 100% Not Observed lugdunensis Streptococcus mitis ATCC 15914100% 100% Not Observed ATCC 49456 100% 100% Not Observed StreptococcusATCC 43078 100% 100% Not Observed dysgalactiae ATCC 35666 100% 100% NotObserved Streptococcus equi ATCC 9528 100% 100% Not ObservedStreptococcus ATCC 49475 100% 100% Not Observed gallolyticus ATCC 9809100% 100% Not Observed Streptococcus infantis ATCC 100% 100% NotObserved 700779 Streptococcus oralis ATCC 35037 100% 100% *Not ObservedStreptococcus ATCC 15909 100% 100% Not Observed parasanguinisStreptococcus salivarius ATCC 25975 100% 100% Not Observed ATCC 7073100% 100% Not Observed Streptococcus ATCC 100% 100% Not Observedthoraltensis 700865 Streptococcus gordonii ATCC 10558 100% 100% NotObserved Streptococcus agalactiae ATCC 12403 100% 100% Not Observed ATCC12973 100% 100% Not Observed ATCC 13813 100% 100% Not ObservedStreptococcus ATCC 6315 100% 100% Not Observed pneumoniae ATCC 6321 100%100% Not Observed ATCC 100% 100% *Not Observed 700673 ATCC 100% 100% NotObserved 700674 ATCC BAA- 100% 100% Not Observed 659 ATCC BAA- 100% 100%Not Observed 1656 ATCC BAA- 100% 100% Not Observed 1667 Streptococcuspyogenes ATCC 14289 100% 100% Not Observed ATCC 19615 100% 100% NotObserved Streptococcus anginosus NCTC 10713 100% 100% Not ObservedStreptococcus ATCC 27513 100% 100% Not Observed constellatusStreptococcus ATCC 27335 100% 100% Not Observed intermedius Candidakrusei ATCC 32196 100% 100% Not Observed Candida parapsilosis ATCC 58895100% 100% Not Observed Acinetobacter baumanii NCTC 13420 100% 100% NotObserved Bacteroides fragilis NCTC 9343 100% 100% Not ObservedCitrobacter freundii NCTC 9750 100% 100% Not Observed Enterobactercloacae ATCC 13047 100% 100% Not Observed Fusobacterium ATCC 25286 100%100% *Not Observed necrophorum Haemophilus influenzae ATCC 4560 100%100% Not Observed Klebsiella pneumoniae ATCC 51503 100% 100% NotObserved Neisseria meningitides ATCC 13113 100% 100% Not Observed(serogroup B) Proteus mirabilis ATCC 43071 100% 100% Not ObservedPseudomonas ATCC 15442 100% 100% Not Observed aeruginosa Salmonellaenterica ATCC 51957 100% 100% Not Observed subsp. enterica Serratiamarcescens ATCC 8100 100% 100% Not Observed *One replicate had alow-level signal for Pan-Candida. Repeat testing of three additionalreplicates showed no false positive signals.

TABLE 13 Cross-reactivity with Targets Not Detected by the BCID-GP Panel(Exclusivity) Highest Concentration Cross-Reactivity Organism StrainTested Result Aspergillus fumigatus ATCC 204305 1 × 10⁷ CFU/mL Notobserved Candida orthopsilosis ATCC 96139 1 × 10⁷ CFU/mL Not observedCryptococcus ATCC 14116 1 × 10⁷ CFU/mL Not observed neoformansRhodotorula minuta ATCC 36236 1 × 10⁷ CFU/mL Not observed SaccharomycesATCC 18824 1 × 10⁷ CFU/mL Not observed cerevisiae Trichosporon asahiiATCC 201110 1 × 10⁷ CFU/mL Not observed Abiotrophia defectiva ATCC 491761 × 10⁹ CFU/mL Not observed Actinomyces ATCC 17929 1 × 10⁹ CFU/mL Notobserved odontolyticus Aerococcus urinae ATCC 700306 1 × 10⁹ CFU/mL Notobserved Aerococcus viridans ATCC 10400 1 × 10⁹ CFU/mL Not observedAnaerococcus prevotii ATCC 9321 1 × 10⁹ CFU/mL Not observedArcanobacterium ATCC BAA- 4.1 × 10⁸ CFU/mL   Not observed haemolyticum1784 Arthrobacter ATCC 700733 1 × 10⁹ CFU/mL Not observedpsychrolactophilus Carnobacterium ATCC 27865 3.6 × 10⁸ CFU/mL   Notobserved maltaromaticum Cellulomonas turbata ATCC 25835 1 × 10⁹ CFU/mLNot observed Clostridium ATCC 25537 1 × 10⁹ CFU/mL Not observedclostridioforme Granulicatella adiacens ATCC 43205 1 × 10⁹ CFU/mL Notobserved Granulicatella elegans ATCC 700633 3.6 × 10⁸ CFU/mL   Notobserved Kocuria kristinae ATCC BAA- 1 × 10⁹ CFU/mL Not observed 752Leuconostoc carnosum ATCC 49367 1 × 10⁹ CFU/mL Not observed Leuconostoccitreum ATCC 13146 1 × 10⁹ CFU/mL Not observed Leuconostoc ATCC 8293 1 ×10⁹ CFU/mL Not observed mesenteroides Macrococcus ATCC 29750 1 × 10⁹CFU/mL Not observed caseolyticus Pediococcus acidilactici ATCC 8042 1 ×10⁹ CFU/mL Not observed Peptostreptococcus ATCC 27337 1 × 10⁹ CFU/mL Notobserved anaerobius Propionibacterium ATCC 11829 1 × 10⁹ CFU/mL Notobserved granulosum Propionibacterium ATCC 14157 1 × 10⁹ CFU/mL Notobserved propionicum Rhodococcus equi ATCC 6939 1 × 10⁹ CFU/mL Notobserved Rothia dentocariosa ATCC 31918 1 × 10⁹ CFU/mL Not observedRothia mucilaginosa ATCC 25296 1 × 10⁹ CFU/mL Not observed

Example 9: Gram-Positive Panel, Competitive Inhibition

Detection of more than one clinically relevant on-panel organism in asample was evaluated with the BCID-GP Panel using eight selectedorganisms which were grouped into mixes of two or three organisms permix in a blood culture matrix. Test case scenarios paired mixes with onemix at approximately ten times the analytically determined limit ofdetection (10×LoD) and a second at high titer (1×10⁸ CFU/mL forbacterial targets and 1×10⁷ CFU/mL for Candida albicans) and vice versa.The organism mixes and combined mixes are summarized in Table 14 andTable 15. All targets were detected in the combinations specified inTable 15 with the exception of mecA in combined mix 2. mecA (carried byS. aureus) was not detected and therefore further tested at 10-foldhigher levels in order to achieve 100% detection. The results ofco-detection testing demonstrate the ability of the BCID-GP to detecttwo on-panel organisms in a sample at both high and low concentrations.

TABLE 14 Detection of Co-Infections: Organism Mixes Organism Mix 1Organism Mix 2 Organism Mix 3 Enterococcus faecium KlebsiellaEnterococcus faecalis (vanA+) pneumoniae (vanB+) Escherichia coliLactobacillus casei Streptococcus pneumoniae Staphylococcus aureusCandida albicans (mecA+)

TABLE 15 Detection of Co-Infections: Organism Mix Pairings Combined MixConcentration ID 10X LoD 1 × 10⁸ CFU/mL* 1 Mix 1 Mix 2 2 Mix 1 Mix 3 3Mix 2 Mix 1 4 Mix 2 Mix 3 5 Mix 3 Mix 1 6 Mix 3 Mix 2 *Candida albicanswas tested at 1 × 10⁷ CFU/mL

Example 10: Gram-Positive Panel, Interfering Substances

Substances

Fifteen substances commonly found in blood culture specimens or asmedications commonly used to treat the skin or blood infections whichcould potentially interfere with the BCID-GP Panel were individuallyevaluated. Each potentially interfering substance was spiked intonegative sample matrix at a medically relevant concentration. Eightorganisms representing 13 targets over a broad range of pathogens on theBCID-GP panel were combined in two mixes to achieve a finalconcentration of 10×LoD each and run in triplicate. No substances testedwere found to inhibit the BCID-GP Panel at the concentrations listed inTable 16. The organisms in the test panel and the interfering substancesare summarized in Tables 16 and 17, respectively.

TABLE 16 Potentially Interfering Substances: Gram-Positive Organism ListMix Target(s) Organism Strain Concentration 1 Enterococcus faecium/Enterococcus faecium ATCC BAA- 1 × 10⁷ CFU/mL vanA (vanA+) 2317Klebsiella pneumoniae Klebsiella pneumoniae ATCC 51503 1 × 10⁷ CFU/mLCandida albicans Candida albicans ATCC 24433 1 × 10⁷ CFU/mLStaphylococcus aureus/ Staphylococcus aureus NRS 70 1 × 10⁶ CFU/mL mecA(mecA+) 2 Enterococcus faecalis/ Enterococcus faecalis ATCC 51575 1 ×10⁷ CFU/mL vanB (vanB+) Streptococcus Streptococcus pneumoniae ATCC BAA-1 × 10⁶ CFU/mL pneumoniae 475 Lactobacillus Lactobacillus casei ATCC 3341 × 10⁶ CFU/mL Staphylococcus Staphylococcus ATCC 49134 1 × 10⁶ CFU/mLepidermidis epidermidis

TABLE 17 Potentially Interfering Substances: Substance List TestingConcentration Endogenous Substances Bilirubin 20 mg/dL Hemoglobin 14 g/LHuman Genomic DNA 6.0 × 10⁴ copies/mL Triglycerides 3000 mg/dLγ-globulin 0.75 g/dL Exogenous Substances Heparin 0.4 U/mLAmoxicillin/Clavulanate 7.5 ug/mL Amphotericin B 2.0 mg/L Ceftriaxone0.152 mg/mL Ciprofloxacin 7.27 mg/L Fluconazole 15.6 mg/L Gentamicinsulfate 0.01 mg/mL Imipenem 0.083 mg/mL Tetracycline 5 mg/L Vancomycin15 mg/L

Bottle Types

The potential inhibitory effect of various blood culture bottles wereevaluated as part of the interfering substance study. A diverse mix offour of organisms that represent eight targets on the BCID-GP panel, wasspiked into sample matrix at a concentration of 10×LoD each based on theanalytically determined limit of detection of the species. Thirteentypes of blood culture bottles were tested in duplicate for each bottletype. One replicate of each bottle type was inoculated with negativeblood only as a negative control. The organisms and bottle types testedare summarized in Table 18 and Table 19, respectively.

All bottle types tested were shown to be compatible with the BCID-GPPanel. None of the bottle types tested were found to inhibit the BCID-GPPanel.

TABLE 18 Potentially Interfering Substances: Bottle Type Gram-PositiveOrganism List Target(s) Evaluated Organism Strain ConcentrationEnterococcus Enterococcus faecium ATCC BAA- 1 × 10⁷ CFU/mL Enterococcusfaecium (vanA+) 2317 vanA Staphylococcus aureus Staphylococcus aureusNRS 70 1 × 10⁶ CFU/mL mecA (mecA+) Klebsiella pneumoniae Klebsiellapneumoniae ATCC 51503 1 × 10⁷ CFU/mL Candida albicans Candida albicansATCC 24433 1 × 10⁷ CFU/mL

TABLE 19 Potentially Interfering Substances: Bottle Types Bottle BrandBottle Type BACTEC Plus Aerobic/F BACTEC Standard/10 Aerobic/F BACTECStandard Anaerobic/F BACTEC Plus Anaerobic/F BACTEC Pediatric PlusBACTEC Lytic/10 Anaerobic/F BacT/ALERT SA Standard Aerobic BacT/ALERT SNStandard Anaerobic BacT/ALERT FA Plus BacT/ALERT FN Plus BacT/ALERT PFPlus VersaTREK REDOX 1 EZ Draw Aerobic VersaTREK REDOX 2 EZ DrawAnaerobic

Example 11, Gram-Negative Blood Culture Contamination

False positives were also observed in the gram-negative panel. As inExample, 1 above, negative blood matrices (no sample, no blood) listedin Table 20 were screened for contaminants. The rubber sealer of eachblood culture bottle was cleaned with ethanol before puncturing it witha needle. 75 uL from each bottle was aspirated. sLRM was performed (BBsample, add 300 uL lysis buffer, 500 uL binding buffer-wait 2 min, andwash with 150 uL wash buffer). Take the washed beads and perform S2A runusing 100% beads.

TABLE 20 Blood Culture Brand Bottle Types BACTEC Plus Aerobic/F BACTECStandard/10 Aerobic/F BACTEC Standard Anaerobic/F BACTEC PlusAnaerobic/F BACTEC Pediatric Plus BACTEC Lytic/10 Anaerobic/F BacT/ALERTSN Standard Anaerobic BacT/ALERT FA Plus BacT/ALERT FN Plus BacT/ALERTPF Plus VersaTREK REDOX 1 EZ Draw Aerobic VersaTREK REDOX 2 EZ DrawAnaerobic

The following organisms were detected as false positives:Stenotrophomonas maltophilia, Klebsiella oxytoca, OXA (OXA-23 andOXA-48), Pseudomonas aeruginosa, Pan Gram-Positive, Enterobactercloacae/hormaechei, Pan Candida, Fusobacterium nucleatum, Escherichiacoli, Serratia, Neisseria meningitides, Citrobacter, Morganellamorganii, Klebsiella penumoniae, Proteus mirabilis, Proteus, Haemophilusinfluenza, Acinetobater baumannii. FIG. 16 shows representative data forthe false positives, Proteus mirabilis (FIG. 16a ), Proteus (FIG. 16b ).

When PCR cycling was reduced from 40 to 30 or 35, no false positiveswere detected.

TABLE 21 BCID-GN reduction in PCR cycle eliminates false positives 40Cycles Reduced Cycles Stenotrophomonas Detected Not detected at 35cycles¹ maltophilia OXA-23 Detected Not detected at 35 cycles² OXA-48Detected Not detected at 30 cycles³ Pan Gram-Positive (7 Detected Notdetected at 30 or 35 cycles, assays) some threshold⁴ Pan CandidaDetected Not detected at 35 cycles Escherichia coli Detected Notdetected at 30 cycles³ Neisseria meningitides Detected Not detected at30 cycles⁷ Morganella morganii Detected Not detected at 30 cycles³Klebsiella penumoniae Detected Not detected at 30 cycles³ Haemophilusinfluenza Detected Not detected at 30 cycles³ Klebsiella oxytocaDetected Not detected at 30 cycles³ Pseudomonas aeruginosa Detected Notdetected at 30 cycles³ Enterobacter Detected Not detected at 30 cycles³cloacae/hormaechei Fusobacterium nucleatum Detected Not detected at 30cycles³ Serratia Detected Not detected at 30 cycles³ CitrobacterDetected Not detected at 30 cycles³ Proteus Detected Not detected at 30cycles³ Proteus mirabilis Detected Not detected at 30 cycles⁵Acinetobater baumannii Detected Not detected at 30 cycles⁶ ¹30 nAboundary set point for target ²50 nA boundary set point for target ³20nA boundary set point for target ⁴Pan Gram-Positive, Enterococcusfaecalis (10 nA); Pan Gram-Positive Bacillus (40 nA); Pan Gram-PositiveStreptococcus anginosus (70 nA); Pan Gram-Positive Enterococcus (15 nA);Pan Gram-PositiveStrep_Staph (70 nA) boundary set point for target ⁵25nA boundary set point for target ⁶10 nA boundary set point for target⁷70 nA boundary set point for target

FIG. 17 shows representative data for the BCID-GN assay with reduced PCRcycling (cycling as indicated in FIG. 18) showing no false positiveswere detected. Specifically, negative (no blood or bacterial targets)BacT/ALERT bottles were tested (˜30 replicates) and the graph in FIG. 17shows only control signals; no contamination.

Example 12: Gram-Negative Panel, Limit of Detection (AnalyticalSensitivity)

The BCID-GN Multiplex primer pool and PCR cycles are shown in FIG. 18.Each of the 8 PCR drops contains an internal control S. pombe is thecontrol target in PCR drops 1-4 (35-cycle PCR) Synthetic Control 1 (SC1)is the control target in PCR drops 5-8 (30-cycle PCR).

The PCR cycling conditions are as follows:

TABLE 22 BCID-GN PCR Cycling Denature Anneal/Extend Cycle No. Hot Start30 sec.  Step 1 3 sec. 27 sec. 1-10 Step 2 3 sec. 42 sec. 11-30 or 11-35

This primer pool and PCR cycling are used for Examples 12-15. TheBCID-GN cartridge layout is shown in FIG. 19 and was also used inExamples 12-15.

The limit of detection (LoD), or analytical sensitivity, was identifiedand verified for each assay on the BCID-GN Panel using quantifiedreference strains. Serial dilutions were prepared in simulated bloodculture sample matrix which is defined as the matrix from a negativeblood culture bottle mixed with whole blood and EDTA in the same ratioas the manufacturer recommends for blood culture. One or more organismsper target were tested, with at least 20 replicates per organism tested.The limit of detection was defined as the lowest concentration of eachtarget that is detected >95% of the time. The confirmed LoD for eachBCID-GN Panel organism is shown in Table 23.

TABLE 23 LoD Results Summary Target Organism Strain LoD ConcentrationAcinetobacter Acinetobacter baumannii NCTC 13421 1 × 10⁶ CFU/mLbaumannii (OXA-23+) Bacteroides fragilis Bacteroides fragilis ATCC 438601 × 10⁴ CFU/mL Citrobacter koseri Citrobacter koseri ATCC 27156 1 × 10⁶CFU/mL Cronobacter Cronobacter sakazakii ATCC 29004 1 × 10⁶ CFU/mLsakazakii Enterobacter non- Enterobacter aerogenes CDC #0074 1 × 10⁵CFU/mL cloacae complex (OXA-48+) Enterobacter amnigenus ATCC 33072 1 ×10⁶ CFU/mL Enterobacter cloacae Enterobacter asburiae ATCC 35957 1 × 10⁶CFU/mL complex Enterobacter cloacae CDC #0154 1 × 10⁶ CFU/mL (VIM+)Escherichia coli Escherichia coli (CTX- NCTC 13441 1 × 10⁶ CFU/mL M+)Fusobacterium Fusobacterium ATCC 51357 1 × 10⁷ CFU/mL necrophorumnecrophorum Fusobacterium Fusobacterium ATCC 25586 1 × 10⁶ CFU/mLnucleatum nucleatum Haemophilus Haemophilus Influenzae ATCC 19418 1 ×10⁶ CFU/mL Influenzae Klebsiella oxytoca Klebsiella oxytoca ATCC 8724 1× 10⁷ CFU/mL Klebsiella Klebsiella pneumoniae ATCC 9436 1 × 10⁶ CFU/mLpneumoniae Morganella morganii Morganella morganii CDC #0133 1 × 10⁶CFU/mL (KPC+) Neisseria Neisseria meningitidis ATCC 13102 1 × 10⁷ CFU/mLmeningitidis Proteus mirabilis Proteus mirabilis CDC #0159 1 × 10⁶CFU/mL (NDM+) Proteus Proteus vulgaris ATCC 6896 1 × 10⁶ CFU/mLPseudomonas Pseudomonas aeruginosa CDC #0103 1 × 10⁷ CFU/mL aeruginosa(IMP+) Salmonella Salmonella bongori ATCC 43975 1 × 10⁵ CFU/mL Serratiamarcescens Serratia marcescens ATCC 14041 1 × 10⁶ CFU/mL SerratiaSerratia plymuthica ATCC 53858 1 × 10⁷ CFU/mL StenotrophomonasStenotrophomonas ATCC 17666 1 × 10⁶ CFU/mL maltophilia maltophilia CTX-MEscherichia coli (CTX- NCTC 13441 1 × 10⁴ CFU/mL M+) IMP Pseudomonasaeruginosa CDC #0103 1 × 10⁵ CFU/mL (IMP+) KPC Morganella morganii CDC#0133 1 × 10⁵ CFU/mL (KPC+) NDM Proteus mirabilis CDC #0159 1 × 10⁵CFU/mL (NDM+) OXA Acinetobacter baumannii NCTC 13421 1 × 10⁵ CFU/mL(OXA-23+) OXA Enterobacter aerogenes CDC #0074 1 × 10⁶ CFU/mL (OXA-48+)VIM Enterobacter cloacae CDC #0154 1 × 10⁵ CFU/mL (VIM+) Pan CandidaCandida albicans ATCC 10231 1 × 10⁵ CFU/mL Candida glabrata ATCC 15126 1× 10⁵ CFU/mL Pan Gram-Positive Bacillus subtilis ATCC 21008 1 × 10⁵CFU/mL Enterococcus faecium ATCC 31282 1 × 10⁷ CFU/mL Staphylococcusaureus ATCC BAA- 1 × 10⁵ CFU/mL 2313 Streptococcus agalactiae ATCC 138131 × 10⁷ CFU/mL

Example 13: Gram-Negative Panel, Analytical Reactivity (Inclusivity andExclusivity)

Analytical Reactivity (Inclusivity)

A panel of 178 strains/isolates representing the genetic, temporal andgeographic diversity of each target on the BCID-GN Panel was evaluatedto demonstrate analytical reactivity. Each bacterial strain was testedin triplicate at 1×10⁸ CFU/mL while each fungus was tested at 1×10⁶CFU/mL in simulated sample matrix.

All of the 178 strains/isolates tested for inclusivity were detected bythe BCID-GN Panel. Results of analytical reactivity are shown in Table24.

Analytical Reactivity (Exclusivity)

Cross-reactivity of on-panel analytes was evaluated using data generatedfrom the Analytical Reactivity study. Cross-reactivity of off-panelorganisms was evaluated by testing a 44 member panel including threeantibiotic resistance markers. Bacterial targets were tested at aconcentration of >1×10⁹ CFU/mL while fungi were tested at aconcentration of >1×10⁷ CFU/mL. If the desired final concentration couldnot be achieved a 2 fold-dilution of the stock organism was used. Table24 summarizes the results of the on-panel organism strains tested. Eachon-panel strain was tested in triplicate. Table 25 summarizes theresults of the off-panel fungal and bacterial strains tested. Nocross-reactivity was observed for any of the off nor on-panel organismswith any of the assays with a few exceptions. Shigella cross-reacts withEscherichia coli due to complete sequence homology as was expected basedon the bioinformatic analysis. Escherichia hermanii may cross-react withat Enterobacter non-cloacae complex at >1×10⁵ CFU/mL and with Serratiaat >1×10⁶ CFU/mL. Acinetobacter anitratus may cross-react withAcinetobacter baumannii at >1×10⁴ CFU/mL.

TABLE 24 Analytical Reactivity (Inclusivity and exclusivity) ResultsHighest Cross- Percent Concentration Reactivity Target Organism StrainDetection Tested Results Acinetobacter Acinetobacter ATCC BAA- 100% 1 ×10⁸ CFU/mL Not baumannii baumannii 1605 observed CDC #0033 100% 1 × 10⁸CFU/mL Not observed NCTC 100% 1 × 10⁸ CFU/mL Not 13421 observed NCTC100% 1 × 10⁸ CFU/mL Not 13424 observed NCTC 100% 1 × 10⁸ CFU/mL Not13304 observed NCTC 100% 1 × 10⁸ CFU/mL Not 13301 observed ATCC BAA-100% 1 × 10⁸ CFU/mL Not 2093 observed NCIMB 100% 1 × 10⁸ CFU/mL Not12457 observed Bacteroides Bacteroides ATCC 9343 100% 1 × 10⁸ CFU/mL Notfragilis fragilis observed ATCC 100% 1 × 10⁸ CFU/mL Not 25285 observedATCC 100% 1 × 10⁸ CFU/mL Not 700786 observed Citrobacter CitrobacterATCC 100% 1 × 10⁸ CFU/mL Not braakii 43162 observed Citrobacter ATCC8090 100% 1 × 10⁸ CFU/mL Not freundii observed CDC #0116 100% 1 × 10⁸CFU/mL Not observed NCTC 8581 100% 1 × 10⁸ CFU/mL Not observed JMI 2047100% 1 × 10⁸ CFU/mL Not observed Citrobacter ATCC 100% 1 × 10⁸ CFU/mLNot koseri 29936 observed ATCC 100% 1 × 10⁸ CFU/mL Not 25409 observedATCC 100% 1 × 10⁸ CFU/mL Not 29225 observed Citrobacter ATCC 100% 1 ×10⁸ CFU/mL Not youngae 29935 observed Citrobacter CDC #0157 100% 1 × 10⁸CFU/mL Not species observed Cronobacter Cronobacter ATCC 100% 1 × 10⁸CFU/mL Not sakazakii sakazakii 29544 observed Enterobacter EnterobacterATCC 100% 1 × 10⁸ CFU/mL Not cloacae asburiae 35957 observed complexATCC 100% 1 × 10⁸ CFU/mL Not 35954 observed Enterobacter NCTC 100% 1 ×10⁸ CFU/mL Not cloacae 13464 observed CDC #0163 100% 1 × 10⁸ CFU/mL Notobserved ATCC 100% 1 × 10⁸ CFU/mL Not 35030 observed Enterobacter ATCC100% 1 × 10⁸ CFU/mL Not hormaechei 49163 observed ATCC 100% 1 × 10⁸CFU/mL Not 700323 observed ATCC BAA- 100% 1 × 10⁸ CFU/mL Not 2082observed Enterobacter Enterobacter ATCC 100% 1 × 10⁸ CFU/mL Notnon-cloacae aerogenes 13048 observed complex ATCC 100% 1 × 10⁸ CFU/mLNot 51697 observed ATCC 100% 1 × 10⁸ CFU/mL Not 29010 observedEnterobacter ATCC 100% 1 × 10⁸ CFU/mL Not amnigenus 51816 observed ATCC100% 1 × 10⁸ CFU/mL Not 33731 observed Enterobacter ATCC 100% 1 × 10⁸CFU/mL Not gergoviae 33426 observed Escherichia Escherichia NCTC 100% 1× 10⁸ CFU/mL Not coli coli 13353 observed NCTC 100% 1 × 10⁸ CFU/mL Not13400 observed NCTC 100% 1 × 10⁸ CFU/mL Not 13452 observed CDC #0118100% 1 × 10⁸ CFU/mL Not observed CDC #0137 100% 1 × 10⁸ CFU/mL Notobserved CDC #0150 100% 1 × 10⁸ CFU/mL Not observed ATCC BAA- 100% 1 ×10⁸ CFU/mL Not 2340 observed LMC_DR00012 100% 1 × 10⁸ CFU/mL Notobserved ATCC 4157 100% 1 × 10⁸ CFU/mL Not observed ATCC 100% 1 × 10⁸CFU/mL Not 51446 observed ATCC 100% 1 × 10⁸ CFU/mL Not 53498 observedATCC 100% 1 × 10⁸ CFU/mL Not 700728 observed ATCC 8545 100% 1 × 10⁸CFU/mL Not observed ATCC 8620 100% 1 × 10⁸ CFU/mL Not observed ATCC 9637100% 1 × 10⁸ CFU/mL Not observed ATCC BAA- 100% 1 × 10⁸ CFU/mL Not 196observed ATCC BAA- 100% 1 × 10⁸ CFU/mL Not 197 observed ATCC BAA- 100% 1× 10⁸ CFU/mL Not 198 observed ATCC BAA- 100% 1 × 10⁸ CFU/mL Not 199observed ATCC BAA- 100% 1 × 10⁸ CFU/mL Not 202 observed ATCC BAA- 100% 1× 10⁸ CFU/mL Not 203 observed ATCC BAA- 100% 1 × 10⁸ CFU/mL Not 204observed ATCC BAA- 100% 1 × 10⁸ CFU/mL Not 201 observed NCTC 100% 1 ×10⁸ CFU/mL Not 13462 observed NCTC 100% 1 × 10⁸ CFU/mL Not 13463observed CDC #0086 100% 1 × 10⁸ CFU/mL Not observed NCTC 100% 1 × 10⁸CFU/mL Not 13450 observed NCTC 100% 1 × 10⁸ CFU/mL Not 13476 observedATCC 100% 1 × 10⁸ CFU/mL Not 13353 observed LMC_243094647 100% 1 × 10⁸CFU/mL Not observed Fusobacterium Fusobacterium ATCC 100% 1 × 10⁸ CFU/mLNot necrophorum necrophorum 27852 observed NCTC 100% 1 × 10⁸ CFU/mL Not10575 observed Fusobacterium Fusobacterium ATCC 100% 1 × 10⁸ CFU/mL Notnucleatum nucleatum 31647 observed ATCC 100% 1 × 10⁸ CFU/mL Not 23726observed Haemophilus Haemophilus ATCC 9332 100% 1 × 10⁸ CFU/mL Notinfluenzae influenzae observed NCTC 8472 100% 1 × 10⁸ CFU/mL Notobserved ATCC 9833 100% 1 × 10⁸ CFU/mL Not observed KlebsiellaKlebsiella ATCC 100% 1 × 10⁸ CFU/mL Not oxytoca oxytoca 13182 observedATCC 100% 1 × 10⁸ CFU/mL Not 43165 observed ATCC 100% 1 × 10⁸ CFU/mL Not43863 observed ATCC 100% 1 × 10⁸ CFU/mL Not 43086 observed KlebsiellaKlebsiella CDC #0112 100% 1 × 10⁸ CFU/mL Not pneumoniae pneumoniaeobserved CDC #0113 100% 1 × 10⁸ CFU/mL Not observed CDC #0125 100% 1 ×10⁸ CFU/mL Not observed ATCC BAA- 100% 1 × 10⁸ CFU/mL Not 1705 observedNCTC 100% 1 × 10⁸ CFU/mL Not 13443 observed CDC #0140 100% 1 × 10⁸CFU/mL Not observed CDC #0141 100% 1 × 10⁸ CFU/mL Not observed NCTC 100%1 × 10⁸ CFU/mL Not 13440 observed NCTC 100% 1 × 10⁸ CFU/mL Not 13439observed IMH- 100% 1 × 10⁸ CFU/mL Not C4171868 observed IMH- 100% 1 ×10⁸ CFU/mL Not C2261309 observed IMH- 100% 1 × 10⁸ CFU/mL Not C3020782observed IMH- 100% 1 × 10⁸ CFU/mL Not C2260742 observed IMH- 100% 1 ×10⁸ CFU/mL Not C4151728 observed ATCC BAA- 100% 1 × 10⁸ CFU/mL Not 1706observed CDC #0075 100% 1 × 10⁸ CFU/mL Not observed CDC #0142 100% 1 ×10⁸ CFU/mL Not observed CDC #0135 100% 1 × 10⁸ CFU/mL Not observed CDC#0153 100% 1 × 10⁸ CFU/mL Not observed CDC #0160 100% 1 × 10⁸ CFU/mL Notobserved Morganella Morganella CDC #0057 100% 1 × 10⁸ CFU/mL Notmorganii morganii observed GM148-209 100% 1 × 10⁸ CFU/mL Not observedATCC 100% 1 × 10⁸ CFU/mL Not 25829 observed Neisseria Neisseria NCTC100% 1 × 10⁸ CFU/mL Not meningitidis meningitidis 10026 observed ATCC100% 1 × 10⁸ CFU/mL Not 13077 observed ATCC 100% 1 × 10⁸ CFU/mL Not35561 observed Proteus/ Proteus CDC #0155 100% 1 × 10⁸ CFU/mL NotProteus mirabilis observed mirabilis ATCC 100% 1 × 10⁸ CFU/mL Not 12453observed ATCC 100% 1 × 10⁸ CFU/mL Not 43071 observed Proteus ProteusATCC 8427 100% 1 × 10⁸ CFU/mL Not vulgaris observed NCTC 4636 100% 1 ×10⁸ CFU/mL Not observed ATCC 100% 1 × 10⁸ CFU/mL Not 49132 observedPseudomonas Pseudomonas CDC #0090 100% 1 × 10⁸ CFU/mL Not aeruginosaaeruginosa observed CDC #0100 100% 1 × 10⁸ CFU/mL Not observed CDC #0054100% 1 × 10⁸ CFU/mL Not observed CDC #0092 100% 1 × 10⁸ CFU/mL Notobserved CDC #0103 100% 1 × 10⁸ CFU/mL Not observed NCTC 100% 1 × 10⁸CFU/mL Not 13437 observed Salmonella Salmonella ATCC 100% 1 × 10⁸ CFU/mLNot enterica 29834 observed serovar Houtenae Salmonella ATCC BAA- 100% 1× 10⁸ CFU/mL Not enterica 1578 observed serovar Indica Salmonella ATCC100% 1 × 10⁸ CFU/mL Not enterica 10721 observed serovar JavianaSalmonella ATCC 9239 100% 1 × 10⁸ CFU/mL Not enterica observed serovarOranienburg Salmonella ATCC 9712 100% 1 × 10⁸ CFU/mL Not entericaobserved serovar Saintpaul Salmonella ATCC 100% 1 × 10⁸ CFU/mL Notenterica 700136 observed serovar Braenderup Salmonella ATCC BAA- 100% 1× 10⁸ CFU/mL Not enterica 708 observed serovar Enteritidis SalmonellaATCC 8391 100% 1 × 10⁸ CFU/mL Not enterica observed serovar ThompsonSalmonella ATCC 9115 100% 1 × 10⁸ CFU/mL Not enterica observed serovarBareilly Salmonella ATCC 8326 100% 1 × 10⁸ CFU/mL Not enterica observedserovar Heidelberg Salmonella ATCC 6962 100% 1 × 10⁸ CFU/mL Not entericaobserved serovar Newport Salmonella FSL A4- 100% 1 × 10⁸ CFU/mL Notenterica 0633 observed serovar Mississippi Serratia/ Serratia ATCC 100%1 × 10⁸ CFU/mL Not Serratia marcescens 43861 observed marcescens ATCC100% 1 × 10⁸ CFU/mL Not 43862 observed ATCC 100% 1 × 10⁸ CFU/mL Not13880 observed Serratia Serratia ATCC 100% 1 × 10⁸ CFU/mL Not plymuthica53858 observed Stenotrophomonas Stenotrophomonas ATCC 100% 1 × 10⁸CFU/mL Not maltophilia maltophilia 13636 observed ATCC 100% 1 × 10⁸CFU/mL Not 13637 observed ATCC 100% 1 × 10⁸ CFU/mL Not 17666 observedPan Candida Candida ATCC 100% 1 × 10⁶ CFU/mL Not albicans 24433 observedCandida ATCC 100% 1 × 10⁶ CFU/mL Not parapsilosis 22019 observed CandidaATCC 100% 1 × 10⁶ CFU/mL Not glabrata 66032 observed Candida krusei ATCC100% 1 × 10⁶ CFU/mL Not 32196 observed Pan Gram- Bacillus cereus ATCC100% 1 × 10⁸ CFU/mL Not Positive 10876 observed Bacillus ATCC 100% 1 ×10⁸ CFU/mL Not atrophaeus 49337 observed Bacillus badius ATCC 100% 1 ×10⁸ CFU/mL Not 14574 observed Bacillus ATCC 100% 1 × 10⁸ CFU/mL Notthuringiensis 35646 observed Bacillus ATCC 100% 1 × 10⁸ CFU/mL Notsubtilis 55614 observed Enterococcus ATCC 100% 1 × 10⁸ CFU/mL Notfaecalis 10100 observed Enterococcus ATCC 100% 1 × 10⁸ CFU/mL Notraffinosus 49464 observed Enterococcus ATCC 100% 1 × 10⁸ CFU/mL Notsaccharolyticus 43076 observed Enterococcus ATCC BAA- 100% 1 × 10⁸CFU/mL Not faecium 2317 observed Enterococcus ATCC 100% 1 × 10⁸ CFU/mLNot casseliflavus 700327 observed Enterococcus ATCC 100% 1 × 10⁸ CFU/mLNot gallinarum 49573 observed Enterococcus ATCC 100% 1 × 10⁸ CFU/mL Notfaecalis 49533 observed Enterococcus ATCC 100% 1 × 10⁸ CFU/mL Notfaecalis 51299 observed Staphylococcus NR-46244 100% 1 × 10⁸ CFU/mL Notaureus observed Staphylococcus ATCC 100% 1 × 10⁸ CFU/mL Not caprae 51548observed Staphylococcus ATCC 100% 1 × 10⁸ CFU/mL Not epidermidis 35984observed Staphylococcus ATCC 100% 1 × 10⁸ CFU/mL Not haemolyticus 29970observed Staphylococcus ATCC 100% 1 × 10⁸ CFU/mL Not lentus 700403observed Staphylococcus ATCC 100% 1 × 10⁸ CFU/mL Not muscae 49910observed Staphylococcus ATCC 100% 1 × 10⁸ CFU/mL Not warneri 27836observed Staphylococcus ATCC 100% 1 × 10⁸ CFU/mL Not arlettae 43957observed Staphylococcus ATCC 100% 1 × 10⁸ CFU/mL Not carnosus 51365observed Staphylococcus ATCC 100% 1 × 10⁸ CFU/mL Not chromogenes 43764observed Staphylococcus ATCC 100% 1 × 10⁸ CFU/mL Not vitulinus 51699observed Staphylococcus ATCC 100% 1 × 10⁸ CFU/mL Not hominis 27844observed Staphylococcus ATCC 100% 1 × 10⁸ CFU/mL Not pseudintermedius49444 observed Staphylococcus ATCC 100% 1 × 10⁸ CFU/mL Not hyicus 11249observed Staphylococcus ATCC 100% 1 × 10⁸ CFU/mL Not saccharolyticus14953 observed Streptococcus ATCC 100% 1 × 10⁸ CFU/mL Not infantis700779 observed Streptococcus ATCC 100% 1 × 10⁸ CFU/mL Not parasanguinis15909 observed Streptococcus ATCC 100% 1 × 10⁸ CFU/mL Not gordonii 35557observed Streptococcus ATCC 100% 1 × 10⁸ CFU/mL Not peroris 700780observed Streptococcus ATCC 100% 1 × 10⁸ CFU/mL Not criceti 19642observed Streptococcus ATCC 9528 100% 1 × 10⁸ CFU/mL Not equi observedStreptococcus ATCC 100% 1 × 10⁸ CFU/mL Not anginosus 33397 observedStreptococcus ATCC 100% 1 × 10⁸ CFU/mL Not agalactiae 13813 observedStreptococcus ATCC 100% 1 × 10⁸ CFU/mL Not bovis 33317 observedStreptococcus ATCC 100% 1 × 10⁸ CFU/mL Not dysgalactiae 35666 observedStreptococcus ATCC 100% 1 × 10⁸ CFU/mL Not equinus 15351 observedStreptococcus ATCC BAA- 100% 1 × 10⁸ CFU/mL Not infantarius 102 observedCTX-M Citrobacter JMI 2047 100% 1 × 10⁸ CFU/mL Not freundii (CTX-observed M+) Enterobacter NCTC 100% 1 × 10⁸ CFU/mL Not cloacae (CTX-13464 observed M+) CDC #0163 100% 1 × 10⁸ CFU/mL Not observedEscherichia NCTC 100% 1 × 10⁸ CFU/mL Not coli (CTX-M+) 13353 observedNCTC 100% 1 × 10⁸ CFU/mL Not 13400 observed NCTC 100% 1 × 10⁸ CFU/mL Not13452 observed NCTC 100% 1 × 10⁸ CFU/mL Not 13462 observed NCTC 100% 1 ×10⁸ CFU/mL Not 13463 observed CDC #0086 100% 1 × 10⁸ CFU/mL Not observedNCTC 100% 1 × 10⁸ CFU/mL Not 13450 observed ATCC 100% 1 × 10⁸ CFU/mL Not13353 observed Klebsiella NCTC 100% 1 × 10⁸ CFU/mL Not pneumoniae 13443observed (CTX-M+) IMP Escherichia NCTC 100% 1 × 10⁸ CFU/mL Not coli(IMP+) 13476 observed Pseudomonas CDC #0092 100% 1 × 10⁸ CFU/mL Notaeruginosa observed (IMP+) CDC #0103 100% 1 × 10⁸ CFU/mL Not observedKPC Citrobacter CDC #0116 100% 1 × 10⁸ CFU/mL Not freundii observed(KPC+) Enterobacter CDC #0163 100% 1 × 10⁸ CFU/mL Not cloacae observed(KPC+) Enterobacter ATCC BAA- 100% 1 × 10⁸ CFU/mL Not hormaechei 2082observed (KPC+) Escherichia ATCC BAA- 100% 1 × 10⁸ CFU/mL Not coli(KPC+) 2340 observed Klebsiella CDC #0112 100% 1 × 10⁸ CFU/mL Notpneumoniae observed (KPC+) CDC #0113 100% 1 × 10⁸ CFU/mL Not observedCDC #0125 100% 1 × 10⁸ CFU/mL Not observed ATCC BAA- 100% 1 × 10⁸ CFU/mLNot 1705 observed Proteus CDC #0155 100% 1 × 10⁸ CFU/mL Not mirabilisobserved (KPC+) Pseudomonas CDC #0090 100% 1 × 10⁸ CFU/mL Not aeruginosaobserved (KPC+) NDM Acinetobacter CDC #0033 100% 1 × 10⁸ CFU/mL Notbaumannii observed (NDM+) Citrobacter CDC #0157 100% 1 × 10⁸ CFU/mL Notspecies observed (NDM+) Escherichia CDC #0118 100% 1 × 10⁸ CFU/mL Notcoli (NDM+) observed CDC #0137 100% 1 × 10⁸ CFU/mL Not observed CDC#0150 100% 1 × 10⁸ CFU/mL Not observed Klebsiella NCTC 100% 1 × 10⁸CFU/mL Not pneumoniae 13443 observed (NDM+) Morganella CDC #0057 100% 1× 10⁸ CFU/mL Not morganii observed (NDM+) Klebsiella CDC #0153 100% 1 ×10⁸ CFU/mL Not pneumoniae observed (NDM+) OXA Acinetobacter ATCC BAA-100% 1 × 10⁸ CFU/mL Not baumannii 1605 observed (OXA+) NCTC 100% 1 × 10⁸CFU/mL Not 13421 observed NCTC 100% 1 × 10⁸ CFU/mL Not 13424 observedNCTC 100% 1 × 10⁸ CFU/mL Not 13304 observed NCTC 100% 1 × 10⁸ CFU/mL Not13301 observed Escherichia LMC_DR00012 100% 1 × 10⁸ CFU/mL Not coli(OXA+) observed Klebsiella CDC #0140 100% 1 × 10⁸ CFU/mL Not pneumoniaeobserved (OXA+) CDC #0141 100% 1 × 10⁸ CFU/mL Not observed CDC #0075100% 1 × 10⁸ CFU/mL Not observed CDC #0142 100% 1 × 10⁸ CFU/mL Notobserved CDC #0153 100% 1 × 10⁸ CFU/mL Not observed CDC #0160 100% 1 ×10⁸ CFU/mL Not observed VIM Klebsiella NCTC 100% 1 × 10⁸ CFU/mL Notpneumoniae 13440 observed (VIM+) NCTC 100% 1 × 10⁸ CFU/mL Not 13439observed Pseudomonas CDC #0100 100% 1 × 10⁸ CFU/mL Not aeruginosaobserved (VIM+) CDC #0054 100% 1 × 10⁸ CFU/mL Not observed NCTC 100% 1 ×10⁸ CFU/mL Not 13437 observed Klebsiella CDC #0135 100% 1 × 10⁸ CFU/mLNot pneumoniae observed (VIM+)

TABLE 25 Cross-reactivity with Organisms Not Detected by the BCID-GNPanel (Exclusivity) Highest Concentration Cross-Reactivity OrganismStrain Tested Results Acinetobacter ATCC 19002 1 × 10⁹ CFU/mL Notobserved haemolyticus Prevotella oralis ATCC 33269 1 × 10⁹ CFU/mL Notobserved Shigella boydii ATCC 9207 1 × 10⁹ CFU/mL Escherichia colidetected Shigella flexneri ATCC 9199 1 × 10⁹ CFU/mL Escherichia colidetected Neisseria sicca ATCC 29193 1 × 10⁹ CFU/mL Not observedNeisseria gonorrhoeae ATCC 19424 1 × 10⁹ CFU/mL Not observed Yersiniaenterocolitica ATCC 9610 1 × 10⁹ CFU/mL Not observed subsp.enterocolitica Yersinia kristensenii ATCC 33639 1 × 10⁹ CFU/mL Notobserved Bacteroides ovatus ATCC BAA-1296 1 × 10⁹ CFU/mL Not observedHaemophilus ATCC 33390 1 × 10⁹ CFU/mL Not observed haemolyticusRalstonia insidiosa ATCC 49129 1 × 10⁹ CFU/mL Not observed Vibrioalginolyticus ATCC 17749 1 × 10⁹ CFU/mL Not observed Vibrio furnissiiNCTC 11218 1 × 10⁹ CFU/mL Not observed Prevotella intermedia ATCC 150321 × 10⁹ CFU/mL Not observed Prevotella corporis ATCC 33547 1 × 10⁹CFU/mL Not observed Pantoea agglomerans ATCC 14537 1 × 10⁹ CFU/mL Notobserved Escherichia hermanii ATCC 700368 1 × 10⁹ CFU/mL Enterobacteramnigenus and Serratia detected^(A) Acinetobacter anitratus ATCC 49139 1× 10⁹ CFU/mL Acinetobacter baumanii detected^(B) Escherichia fergusoniiATCC 35469 1 × 10⁹ CFU/mL Not observed Bacteroides merdae ATCC 43184 1 ×10⁹ CFU/mL Not observed Bacteroides distasonis ATCC 8503 1 × 10⁹ CFU/mLNot observed (Parabacteroides) Bacteroides eggerthii ATCC 27754 1 × 10⁹CFU/mL Not observed Prevotella nigrescens ATCC 33563 1 × 10⁹ CFU/mL Notobserved Bordetella pertussis ATCC 9797 1 × 10⁹ CFU/mL Not observedPseudomonas mosselii ATCC 49838 1 × 10⁹ CFU/mL Not observed Pseudomonasfluorescens ATCC 13525 1 × 10⁹ CFU/mL Not observed Neisseria flavescensATCC 13115 1 × 10⁹ CFU/mL Not observed Pasteurella aerogenes ATCC 278831 × 10⁹ CFU/mL Not observed Providencia alcalifaciens ATCC 9886 1 × 10⁹CFU/mL Not observed Escherichia coli (TEM) CTC 13351 1 × 10⁹ CFU/mL Notobserved^(C) Klebsiella pneumoniae CDC# 0087 1 × 10⁹ CFU/mL Notobserved^(C) (SHV) Serratia marcescens CDC# 0091 1 × 10⁹ CFU/mL Notobserved^(C) (SME) Lactococcus lactis ATCC 49032 1 × 10⁹ CFU/mL Notobserved Lactobacillus acidophilus ATCC 314 1 × 10⁹ CFU/mL Not observedCorynebacterium renale ATCC 19412 1 × 10⁹ CFU/mL Not observedCorynebacterium ATCCBAA-949 1 × 10⁹ CFU/mL Not observed jeikeiumCorynebacterium ATCC 13812 1 × 10⁹ CFU/mL Not observed diphtheriaeListeria innocua ATCC 33090 1 × 10⁹ CFU/mL Not observed Lactobacilluscasei ATCC 39392 1 × 10⁹ CFU/mL Not observed Micrococcus luteus ATCC10240 1 × 10⁹ CFU/mL Not observed Corynebacterium ATCC 51799 1 × 10⁹CFU/mL Not observed ulcerans Candida tropicalis ATCC 1369 1 × 10⁷ CFU/mLNot observed Candida orthopsilosis ATCC 96139 1 × 10⁷ CFU/mL Notobserved Trichosporon asahii ATCC 201110 1 × 10⁷ CFU/mL Not observedPrevotella intermedia ATCC 15032 9 × 10⁸ CFU/mL Not observed Prevotellacorporis ATCC 33547 6 × 10⁸ CFU/mL Not observed Pseudomonas mosseliiATCC 49838 1 × 10⁹ CFU/mL Not observed Prevotella oralis ATCC 33269 5 ×10⁸ CFU/mL Not observed Bacteroides ovatus ATCC BAA-1296 6 × 10⁸ CFU/mLNot observed Haemophilus ATCC 33390 4 × 10⁸ CFU/mL Not observedhaemolyticus Prevotella nigrescens ATCC 33563 4 × 10⁸ CFU/mL Notobserved Lactobacillus acidophilus ATCC 314 3 × 10⁸ CFU/mL Not observedCorynebacterium ATCC 13812 5 × 10⁸ CFU/mL Not observed diphtheriae ^(A)Enterobacter amnigenus detected at >1 × 10⁵ CFU/mL, Serratia detectedat >1 × 10⁶ CFU/mL ^(B) Acinetobacter baumanii detected at >1 × 10⁴CFU/mL ^(C)Cross-reactivity was not observed for the resistance marker.The on-panel organism was detected as expected.

Example 14: Gram-Negative Panel Competitive Inhibition

Detection of more than one clinically relevant on-panel organism in asample was evaluated with the BCID-GN Panel using nine selectedorganisms which were grouped into mixes of three organisms per mix in ablood culture matrix. Test case scenarios paired mixes with one mix atapproximately ten times the analytically determined limit of detectionfor the species (10×LoD) and a second at high titer (1×10⁸ CFU/mL forbacterial targets and 1×10⁷ CFU/mL for Candida albicans) and vice versa.The organism mixes and combined mixes are summarized in Table 26 andTable 27. All targets were detected in the combinations specified inTable 27. The results of co-detection testing demonstrate the ability ofthe BCID-GN to detect two on-panel organisms in a sample at both highand low concentrations.

TABLE 26 Detection of Co-Infections: Organism Mixes Organism MixTarget(s) Organism Strain 1 Pan Candida Candida albicans ATCC 10231Escherichia coli/ Escherichia coli NCTC CTX-M (CTX-M+) 13441 PanGram-Positive Staphylococcus aureus ATCC BAA- 2313 2 Enterobactercloacae Enterobacter cloacae CDC #0154 complex/VIM (VIM+) Klebsiellapneumoniae Klebsiella pneumoniae ATCC 9436 Serratia/Serratia Serratiamarcescens ATCC marcescens 14041 3 Klebsiella oxytoca Klebsiella oxytocaATCC 8724 Proteus/Proteus Proteus mirabilis CDC #0159 mirabilis/NDM(NDM+) Pseudomonas Pseudomonas CDC #0103 aeruginosa aeruginosa

TABLE 27 Detection of Co-Infections: Organism Mix Pairings Combined MixConcentration ID 10X LoD 1 × 10⁸ CFU/mL* 1 Mix 1 Mix 2 2 Mix 1 Mix 3 3Mix 2 Mix 1 4 Mix 2 Mix 3 5 Mix 3 Mix 1 6 Mix 3 Mix 2 *Candida albicanswas tested at 1 × 10⁷ CFU/mL.

Example 15: Interfering Substances

Substances

Fifteen substances commonly found in blood culture specimens or asmedications commonly used to treat the skin or blood infections whichcould potentially interfere with the BCID-GN Panel were individuallyevaluated. Each potentially interfering substance was spiked intonegative sample matrix at a medically relevant concentration. Sixorganisms representing 9 targets covering a broad range of pathogens onthe BCID-GN Panel were spiked into negative blood matrix to achieve afinal concentration of 10×LoD for each species and run in triplicate. Nosubstances tested were found to inhibit the BCID-GN Panel at theorganism concentrations listed in Table 28 and the substanceconcentrations listed in Table 29.

TABLE 28 Potentially Interfering Substances: Gram-Negative Organism ListTarget(s) Organism Strain Concentration Enterobacter cloacae/Enterobacter cloacae CDC #0154 1 × 10⁷ CFU/mL VIM (VIM+) Escherichiacoli/CTX-M Escherichia coli (CTX- NCTC 13441 1 × 10⁷ CFU/mL M+)Klebsiella pneumoniae Klebsiella pneumoniae ATCC 9436 1 × 10⁷ CFU/mLSerratia/Serratia Serratia marcescens ATCC 14041 1 × 10⁷ CFU/mLmarcescens Pan Candida Candida glabrata ATCC 15126 1 × 10⁶ CFU/mL PanGram-Positive Staphylococcus aureus ATCC BAA- 1 × 10⁶ CFU/mL 2313

TABLE 29 Potentially Interfering Substances: Substance List TestingConcentration Endogenous Substances Bilirubin 20 mg/dL Hemoglobin 14 g/LHuman Genomic DNA 6.0 × 10⁴ copies/mL Triglycerides 3000 mg/dLγ-globulin 0.75 g/dL Exogenous Substances Heparin 0.4 U/mLAmoxicillin/Clavulanate 7.5 ug/mL Amphotericin B 2.0 mg/L Ceftriaxone0.152 mg/mL Ciprofloxacin 7.27 mg/L Fluconazole 15.6 mg/L Gentamicinsulfate 0.01 mg/mL Imipenem 0.083 mg/mL Tetracycline 5 mg/L Vancomycin15 mg/L

Bottle Types

The potential inhibitory effect of various blood culture bottles wereevaluated as part of the interfering substance study. Six organismsrepresenting 9 targets covering a broad range of pathogens on theBCID-GN Panel, were spiked into sample matrix at a concentration of10×LoD each based on the analytically determined limit of detection ofthe species. Thirteen types of blood culture bottles were tested induplicate for each bottle type. One replicate of each bottle type wasinoculated with negative blood only as a negative control. The organismsand bottle types tested are summarized in Table 30 and Table 31,respectively.

All bottle types tested were shown to be compatible with the BCID-GNPanel. None of the bottle types tested were found to inhibit the BCID-GNPanel.

TABLE 30 Potentially Interfering Substances: Bottle Type Gram-NegativeOrganism List Target(s) Organism Strain Concentration Escherichiacoli/CTX-M Escherichia coli (CTX- NCTC 13441 1 × 10⁷ CFU/mL M+)Enterobacter cloacae Enterobacter cloacae CDC #0154 1 × 10⁷ CFU/mLcomplex/VIM (VIM+) Klebsiella pneumoniae Klebsiella pneumoniae ATCC 94361 × 10⁷ CFU/mL Serratia/Serratia Serratia marcescens ATCC 14041 1 × 10⁷CFU/mL marcescens Pan Candida Candida glabrata ATCC 15126 1 × 10⁶ CFU/mLPan Gram-Positive Staphylococcus aureus ATCC BAA- 1 × 10⁶ CFU/mL 2313

TABLE 31 Potentially Interfering Substances: Bottle Types Bottle BrandBottle Type BACTEC Plus Aerobic/F BACTEC Standard/10 Aerobic/F BACTECStandard Anaerobic/F BACTEC Plus Anaerobic/F BACTEC Pediatric PlusBACTEC Lytic/10 Anaerobic/F BacT/ALERT SA Standard Aerobic BacT/ALERT SNStandard Anaerobic BacT/ALERT FA Plus BacT/ALERT FN Plus BacT/ALERT PFPlus VersaTREK REDOX 1 EZ Draw Aerobic VersaTREK REDOX 2 EZ DrawAnaerobic

Example 16, Fungal Blood Culture Contamination

False positives were also observed in the BCID-FP panel.

Unlike for the BCID-GP and GN panels, false positives werereduced/eliminated in the BCID-FP panel by introducing mismatchedprimers.

Table 32 shows the percentage of false positives obtained beforemismatching and thresholding and the percentage of false positives aftermismatching and thresholding. FIG. 20 shows the signals obtained beforeand after detuning, i.e., before and after mismatching and thresholdingfor two targets Rhodotorula (FIG. 20a ) and Trichosporon (FIG. 20b ).

TABLE 32 BCID-FP, Percentage false positives before detuning and afterdetuning False positives False positives after Assay before mismatch,mismatch, (threshold) threshold thresholding Rhodotorula 30/782 (3.8%)2/1254 (0.16%) (≥50 nA) Trichosporon 11/2042 (0.54%) 1/1371 (0.07%) (≥10nA)

Example 17, Fungal Panel, Two Zone, One Target

An additional approach to overcome false positives was applied to twotargets of the BCID-FP assay. Rather than changing the PCR cycle numberor primer concentrations, primers to amplify a single target were placedin two multiplex primer pools. The target is amplified in two PCR lanesand is detected in two different detection zones. A positive detectioncall is only made when the target is detected in both detection zones.If the target is detected in one zone but not the other, a non-detectioncall is made. Without dual zone detection, low level contamination maytrigger amplification and detection at low frequencies. With dual zonedetection, which requires two amplifications and detections, thefrequency of background contamination detection is reduced.

Table 33 shows the percentage of false positive before dual zonedetection and after dual zone detection.

TABLE 33 BCID-FP Dual Zone detection % False Positives Before % FalsePositives Target Dual Zone After Dual Zone C. parapsilosis 0.4% (4/926)0.1% (2/1826) C. tropicalis 0.6% (8/1280) 0.1% (2/1877)

Table 34 shows which targets were falsely detected before detuning(primer mismatch or dual zone detection and thresholding) and after aswell as the thresholding for each target.

TABLE 34 BCID-FP, Detection of false positives before detuning and afterdetuning After Threshold Target Name Before Detuning Detuning (nA)Candida auris Not Detected Not 5 Detected Candida albicans Not DetectedNot 5 Detected Candida dubliniensis Not Detected Not 25 Detected Candidafamata Not Detected Not 100 Detected Candida glabrata Not Detected Not25 Detected Candida guilliermondii Not Detected Not 25 Detected Candidakefyr Not Detected Not 25 Detected Candida krusei Not Detected Not 25Detected Candida lusitaniae Not Detected Not 25 Detected Candidaparapsilosis ² Detected Not 25 Detected Candida tropicalis ² DetectedNot 25 Detected Cryptococcus gattii Not Detected Not 25 DetectedCryptococcus neoformans Not Detected Not 25 var. grubii DetectedCryptococcus neoformans var. Not Detected Not 25 neoformans DetectedFusarium Not Detected Not 25 Detected Malassezia furfur Not Detected Not400 Detected Rhodotorula ¹ Detected Not 50 Detected Trichosporon ¹Detected Not 10 Detected ¹With primer mismatch. ²With dual zonedetection

Example 18, Fungal Panel, Limit of Detection (Analytical Sensitivity)

The BCID-FP Multiplex primer pool and PCR cycles are shown in FIG. 21.S. pombe is the control target in PCR drops 1-4 (40-cycle PCR).

The PCR cycling conditions are as follows:

TABLE 35 BCID-FP PCR cycling Denature 96.3° C. Anneal/Extend 62.5° C.Cycle No. Hot Start 30 Step 1 3 sec 27 sec  1-15 Step 2 3 sec 40 sec16-40

This primer pool and PCR cycling are used for Examples 18-21. TheBCID-FP cartridge layout is shown in FIG. 22 and was also used inExamples 18-21 below.

The limit of detection (LoD) or analytical sensitivity was identifiedand verified for each fungal target on the BCID-FP Panel usingquantified reference strains. Serial dilutions were prepared insimulated blood culture sample matrix (sample matrix), which is definedas the matrix from a negative blood culture bottle mixed with wholeblood and EDTA in the same ratio as the manufacturer recommends forblood culture. Organisms were tested with at least 20 replicates splitbetween two cartridge lots. The limit of detection was defined as thelowest concentration of each target that is detected >95% of the time.The confirmed LoD for each BCID-FP Panel organism is shown in Table 36.

TABLE 36 LoD Results Summary Target Organism Strain LoD ConcentrationCandida albicans Candida albicans ATCC 14053 1.0 × 10⁴ CFU/mL CandidaCandida dubliniensis ATCC MYA-577 3.0 × 10⁴ CFU/mL dubliniensis Candidafamata Candida famata CBS 767 3.0 × 10³ CFU/mL Candida glabrata Candidaglabrata ATCC 2001 1.0 × 10⁴ CFU/mL Candida Candida guilliermondii ATCC22017 1.0 × 10⁴ CFU/mL guilliermondii Candida kefyr Candida kefyr ATCC4135 2.0 × 10² CFU/mL Candida Candida lusitaniae ATCC 34449 1.0 × 10⁴CFU/mL lusitaniae Candid krusei Candid krusei ATCC 22985 2.0 × 10⁴CFU/mL Candida Candida parapsilosis ATCC 28475 3.0 × 10⁴ CFU/mLparapsilosis Candida tropicalis Candida tropicalis ATCC 13803 5.0 × 10⁴CFU/mL Cryptococcus Cryptococcus ATCC 208821 3.0 × 10³ CFU/mL neoformansneoformans var grubii Cryptococcus Cryptococcus gattii ATCC MYA- 1.0 ×10³ CFU/mL gattii 4877 Fusarium Fusarium verticillioides CBS 100312 3.0× 10⁷ CFU/mL Malassezia furfur Malassezia furfur CBS 7710 3.0 × 10⁴CFU/mL Rhodotorula Rhodotorula ATCC 4058 3.0 × 10³ CFU/mL mucilaginosaTrichosporon Trichosporon dermatis ATCC 204094 1.0 × 10⁵ CFU/mL

Example 19, Fungal Panel, Analytical Reactivity (Inclusivity,Cross-Reactivity and Exclusivity)

Analytical reactivity (inclusivity) for the BCID-FP Panel was evaluatedusing a collection of 48 fungal isolates covering the genetic diversityof the organisms detected on the BCID-FP Panel. Negative sample matrixwas spiked with the organism at a concentration of 10×LoD, with a totalof 3 replicates tested for each isolate. The results of the BCID-FPPanel Analytical Reactivity (Inclusivity) Study is shown in Table 37.Cross-Reactivity and Exclusivity

Cross-reactivity of on-panel analytes was evaluated using data generatedfrom the Analytical Reactivity study. Cross-reactivity of off-panelorganisms was evaluated by testing a 36 member panel, containingclinically-relevant bacteria and fungi. Bacterial targets were tested ata concentration of ≥1×109 CFU/mL while fungi were tested at aconcentration of >1×107 CFU/mL whenever possible. Table 38 summarizesthe results of the on-panel fungal strains tested. Each on-panel strainwas tested in triplicate. Table 39 summarizes the results of theoff-panel fungal and bacterial strains tested. No cross-reactivity wasobserved for any of the off- or on-panel organisms.

TABLE 37 Analytical Reactivity (Inclusivity, Cross-Reactivity, andExclusivity) Results Multiple of LoD Organism Strain ConcentrationDetected Candida albicans ATCC MYA-4441 1.0 × 10⁵ CFU/mL 10x Candidaalbicans ATCC 90028 1.0 × 10⁵ CFU/mL 10x Candida NCPF 3949 3.0 × 10⁵CFU/mL 10x dubliniensis Candida ATCC MYA-582 3.0 × 10⁵ CFU/mL 10xdubliniensis Candida famata CBS 1961 3.0 × 10⁴ CFU/mL 10x Candida famataCBS 766 3.0 × 10⁴ CFU/mL 10x Candida glabrata ATCC MYA-2950 1.0 × 10⁵CFU/mL 10x Candida glabrata ATCC 15545 1.0 × 10⁵ CFU/mL 10x Candida ATCC6260 1.0 × 10⁵ CFU/mL 10x guilliermondii Candida ATCC 90197 1.0 × 10⁵CFU/mL 10x guilliermondii Candida kefyr ATCC 204093 2.0 × 10³ CFU/mL 10xCandida kefyr ATCC 8553 2.0 × 10³ CFU/mL 10x Candida krusei ATCC 288702.0 × 10⁵ CFU/mL 10x Candida krusei ATCC 14243 2.0 × 10⁵ CFU/mL 10xCandid lusitaniae ATCC 42720 1.0 × 10⁵ CFU/mL 10x Candid lusitaniae ATCC66035 1.0 × 10⁵ CFU/mL 10x Candida ATCC 28474 3.0 × 10⁵ CFU/mL 10xparapsilosis Candida ATCC 22019 3.0 × 10⁵ CFU/mL 10x parapsilosisCandida ATCC 201381 5.0 × 10⁵ CFU/mL 10x tropicalis Candida ATCC 13695.0 × 10⁵ CFU/mL 10x tropicalis Cryptococcus ATCC MYA-4138 1.0 × 10⁴CFU/mL 10x gattii Cryptococcus ATCC 4560 1.0 × 10⁴ CFU/mL 10x gattiiCryptococcus ATCC MYA-565 3.0 × 10⁴ CFU/mL 10x neoformans CryptococcusATCC 14116 3.0 × 10⁴ CFU/mL 10x neoformans Fusarium CBS 116611 3.0 × 10⁵CFU/mL 10x oxysporum Fusarium CBS 119828 3.0 × 10⁵ CFU/mL 10x sacchariMalassezia furfur ATCC 14521 3.0 × 10⁵ CFU/mL 10x Malassezia furfur ATCC44345 3.0 × 10⁵ CFU/mL 10x Rhodotorula ATCC 4058 3.0 × 10⁴ CFU/mL 10xmucilaginosa Rhodotorula ATCC 96365 3.0 × 10⁴ CFU/mL 10x glutinisTrichosporon ATCC 201110 1.0 × 10⁶ CFU/mL 10x asahii Trichosporon ATCC90043 1.0 × 10⁶ CFU/mL 10x asteroides

TABLE 38 Cross-reactivity with BCID-FP Panel On-Panel Organisms HighestCross- Concentration Reactivity Organism Strain Tested Results Candidaalbicans ATCC 14053 1.0 × 10⁷ CFU/mL Not observed Candida dubliniensisATCC MYA-577 1.0 × 10⁷ CFU/mL Not observed Candida dubliniensis ATCCMYA-578 1.0 × 10⁷ CFU/mL Not observed Candida dubliniensis ATCC MYA-5821.0 × 10⁷ CFU/mL Not observed Candida famata CBS 767 1.0 × 10⁷ CFU/mLNot observed Candida glabrata ATCC 2001 1.0 × 10⁷ CFU/mL Not observedCandida guilliermondii ATCC 22017 1.0 × 10⁷ CFU/mL Not observed Candidakefyr ATCC 4135 1.0 × 10⁷ CFU/mL Not observed Candida lusitaniae ATCC34449 1.0 × 10⁷ CFU/mL Not observed Candid krusei ATCC 22985 1.0 × 10⁷CFU/mL Not observed Candida parapsilosis ATCC 28475 1.0 × 10⁷ CFU/mL Notobserved Candida tropicalis ATCC 13803 1.0 × 10⁷ CFU/mL Not observedCryptococcus gattii ATCC MYA-4877 1.0 × 10⁷ CFU/mL Not observedCryptococcus neoformans ATCC 208821 1.0 × 10⁷ CFU/mL Not observedFusarium verticillioides ATCC 100312 1.0 × 10⁷ CFU/mL Not observedMalassezia furfur CBS 7710 1.0 × 10⁷ CFU/mL Not observed Rhodotorulamucilaginosa ATCC 4058 1.0 × 10⁷ CFU/mL Not observed Trichosporondermatis ATCC 204094 1.0 × 10⁷ CFU/mL Not observed

TABLE 39 Cross-reactivity with Organisms Not Detected by the BCID-FPPanel (Exclusivity) Highest Cross- Concentration Reactivity OrganismClassification Strain Tested Results Aspergillus fumigatus Fungus ATCC2.6 × 10⁶ CFU/mL Not 204305 observed Candida bracarensis Fungus CBS10154 1.0 × 10⁷ CFU/mL Not observed Candida metapsilosis Fungus ATCC96144 1.0 × 10⁷ CFU/mL Not observed Candida orthopsilosis Fungus ATCC96139 1.0 × 10⁷ CFU/mL Not observed Candida rugosa Fungus CBS 96275 1.0× 10⁷ CFU/mL Not observed Filobasidium elegans Fungus CBS 7637 1.0 × 10⁷CFU/mL Not observed Filobasidium Fungus CBS 7642 1.0 × 10⁷ CFU/mL Notglobisporum observed Kluyveromyces lactis Fungus ATCC 10689 1.0 × 10⁷CFU/mL Not observed Malassezia globosa Fungus ATCC 1.0 × 10⁷ CFU/mL NotMYA-4612 observed Malassezia restricta Fungus ATCC 1.0 × 10⁷ CFU/mL NotMYA-4611 observed Malassezia sympodialis Fungus ATCC 44031 1.0 × 10⁷CFU/mL Not observed Saccharomyces Fungus ATCC 18824 1.0 × 10⁷ CFU/mL Notcerevisiae observed Schizosaccharomyces Fungus LPY 02387 4.9 × 10⁶CFU/mL Not pombe observed Sporidiobolus Fungus ATCC 24217 1.0 × 10⁷CFU/mL Not salmonicolor observed Acinetobacter lwoffii Gram- ATCC 153091.0 × 10⁹ CFU/mL Not negative observed Bacteroides fragilis Gram- ATCC25285 1.0 × 10⁹ CFU/mL Not negative observed Bordetella pertussis Gram-ATCC 9340 1.0 × 10⁹ CFU/mL Not negative observed Citrobacter freundiiGram- ATCC 6879 1.0 × 10⁹ CFU/mL Not negative observed Enterobacteraerogenes Gram- ATCC 29751 3.5 × 10⁸ CFU/mL Not negative observedEnterobacter cloacae Gram- ATCC 23373 1.0 × 10⁹ CFU/mL Not negativeobserved Klebsiella oxytoca Gram- ATCC 43165 1.0 × 10⁹ CFU/mL Notnegative observed Morganella morganii Gram- ATCC 25830 1.0 × 10⁹ CFU/mLNot negative observed Proteus mirabilis Gram- ATCC 35659 1.0 × 10⁹CFU/mL Not negative observed Salmonella enterica Gram- ATCC 19430 1.0 ×10⁹ CFU/mL Not Typhi negative observed Serratia marcescens Gram- ATCC43861 1.0 × 10⁹ CFU/mL Not negative observed Clostridium perfringensGram-positive ATCC 13124 1.0 × 10⁹ CFU/mL Not observed CorynebacteriumGram-positive ATCC 7094 1.0 × 10⁹ CFU/mL Not striatum observedEnterococcus faecium Gram-positive ATCC 31282 1.0 × 10⁹ CFU/mL Notobserved Lactobacillus Gram-positive ATCC 53103 1.0 × 10⁹ CFU/mL Notrhamnosus observed Micrococcus luteus Gram-positive ATCC 19212 1.0 × 10⁹CFU/mL Not observed Staphylococcus hominis Gram-positive ATCC 27844 1.0× 10⁹ CFU/mL Not (CoNS) observed Staphylococcus Gram-positive ATCC 296631.0 × 10⁹ CFU/mL Not intermedius (CoPS) observed StaphylococcusGram-positive ATCC 15305 1.0 × 10⁹ CFU/mL Not saprophyticus (CoNS)observed Streptococcus Gram-positive ATCC 12401 1.0 × 10⁹ CFU/mL Notagalactiae (Group B) observed Streptococcus anginosus Gram-positive ATCC9895 1.0 × 10⁹ CFU/mL Not (Group F) observed Streptococcus pyogenesGram-positive ATCC 12384 1.0 × 10⁹ CFU/mL Not (Group A) observed

Example 20, Fungal Panel, Competitive Inhibition

Detection of more than one clinically relevant fungal organism in asample was evaluated with the BCID-FP Panel using sample matrix spikedwith Candida albicans paired with Candida glabrata or Candidaparapsilosis. Candida albicans was tested at 1×10⁷ CFU/mL in conjunctionwith the other two Candida species at 10×LoD, and both Candida glabrataand Candida parapsilosis were tested at 1×10⁷ CFU/mL in combination withCandida albicans at 10×LoD. Additionally, Candida albicans was tested at10×LoD in combination with nine off-panel bacteria each at >1×10⁹CFU/mL. If Candida albicans was not detected in triplicate at 10×LoD inthe presence of any off-panel bacteria, testing was repeated at 30×LoD.These results, summarized in Table 40, demonstrate the ability of theBCID-FP Panel to detect two organisms in a sample at both high and lowconcentrations as well as the ability to detect low concentrations ofclinically relevant fungi in the presence of a high concentration ofoff-panel organism.

TABLE 40 Detection of Co-Infections Results Organism 1 Organism 2Organism 1/ Organism 1 Concentration Organism 2 Concentration Organism 2Candida 10x LoD Candida glabrata ¹ 1.0 × 10⁷ CFU/mL Positive/Positivealbicans ¹ Candida 10x LOD Candida parapsilosis ¹ 1.0 × 10⁷ CFU/mLPositive/Positive albicans ¹ Candida 10x LOD Candida albicans ¹ 1.0 ×10⁷ CFU/mL Positive/Positive glabrata ¹ Candida 10x LOD Candida albicans¹ 1.0 × 10⁷ CFU/mL Positive/Positive parapsilosis ¹ Candida 30x LoDAcinetobacter 1.0 × 10⁹ CFU/mL Positive/N/A albicans ¹ baumannii Candida10x LoD Enterococcus faecalis 1.0 × 10⁹ CFU/mL Positive/N/A albicans ¹Candida 10x LoD Escherichia coli 1.0 × 10⁹ CFU/mL Positive/N/A albicans¹ Candida 10x LoD Klebsiella pneumoniae 1.0 × 10⁹ CFU/mL Positive/N/Aalbicans ¹ Candida 10x LoD Pseudomonas 1.0 × 10⁹ CFU/mL Positive/N/Aalbicans ¹ aeruginosa Candida 10x LoD Staphylococcus aureus 1.0 × 10⁹CFU/mL Positive/N/A albicans ¹ Candida 10x LoD Staphylococcus 1.0 × 10⁹CFU/mL Positive/N/A albicans ¹ epidermidis Candida 10x LoD Streptococcus1.0 × 10⁹ CFU/mL Positive/N/A albicans ¹ pneumoniae Candida 10x LoDPropionibacterium 1.0 × 10⁹ CFU/mL Positive/N/A albicans ¹ acnes¹On-panel organism

Example 21, Fungal Panel, Interfering Substances

Substances

Fifteen substances commonly found in blood culture specimens or asmedications commonly used to treat the skin or bloodstream infectionswhich could potentially interfere with the BCID-FP Panel wereindividually evaluated. Each potentially interfering substance wasspiked into negative sample matrix at a medically relevantconcentration. Five organisms representing a broad range of fungalpathogens were combined to achieve a final concentration of 10× each andrun in triplicate. The organisms on the test panel and the interferingsubstances are summarized in Tables 42 and 43, respectively.

Blood Culture Bottles

The potential inhibitory effect of various blood culture bottle typeswere evaluated as part of the interfering substance study. A diversesub-panel containing 5 fungi, including the most prevalent fungalorganisms identified in positive blood culture, was spiked into samplematrix at a concentration of 10×LoD each, in fifteen types of bloodculture bottles. Two replicates were tested per bottle type. Onereplicate of each bottle type was inoculated with negative blood only asa negative control. The study is summarized in Table 41.

All substances and organisms tested for interference were shown to becompatible with the BCID-FP Panel. No potentially interfering substancesor bottle types were found to inhibit the BCID-FP Panel at theconcentrations tested.

TABLE 41 Potentially Interfering Substances: Fungal Organism List AssayEvaluated Organism Strain Concentration Candida kefyr Candida kefyr ATCC10x LoD 4135 Cryptococcus Cryptococcus ATCC 10x LoD neoformansneoformans grubii 208821 Fusarium Fusarium verticillioides CBS 10x LoD100312 Rhodotorula Rhodotorula ATCC 10x LoD mucilaginosa 4058 Candidaalbicans Candida albicans ATCC 10x LoD 14053

TABLE 42 Potentially Interfering Substances: Substance List TestingConcentration Endogenous Substances Bilirubin 20 mg/dL Hemoglobin 14 g/LHuman Genomic DNA 6.0 × 10⁴ copies/mL Triglycerides 3000 mg/dLγ-globulin 5.4 g/dL Exogenous Substances Heparin 0.4 U/mLAmoxicillin/Clavulanate 7.5 ug/mL Amphotericin B 2.0 mg/L Ceftriaxone0.152 mg/mL Ciprofloxacin 7.27 mg/L Fluconazole 15.6 mg/L Gentamicinsulfate 0.01 mg/mL Imipenem 0.083 mg/mL Tetracycline 5 mg/L Vancomycin15 mg/L

TABLE 43 Potentially Interfering Substances: Bottle Types Bottle BrandBottle Type BACTEC Plus Aerobic/F BACTEC Standard/10 Aerobic/F BACTECStandard Anaerobic/F BACTEC Plus Anaerobic/F BACTEC Pediatric PlusBACTEC Lytic/10 Anaerobic/F BACTEC MYCO/F Lytic BacT/ALERT SA StandardAerobic BacT/ALERT SN Standard Anaerobic BacT/ALERT FA Aerobic FANBacT/ALERT FN Anaerobic FAN BacT/ALERT PF Pediatric FAN BacT/ALERT MPMycobacteria for yeast/fungi VersaTREK REDOX 1 EZ Draw Aerobic VersaTREKREDOX 2 EZ Draw Anaerobic

1. An in vitro method for identifying a first microorganism and secondmicroorganism that infects a subject comprising: (a) loading a portionof a sample comprising (i) reagents for amplifying pan-targets forgram-negative bacteria, species or genus targets for gram-positivebacteria, and species targets for fungi or (ii) reagents for amplifyingspecies or genus targets for gram-positive bacteria, species or genustargets for gram-negative bacteria and species targets for fungi; (b)amplifying nucleic acid in the portion of the sample using the reagentsin step (a) in the first cartridge to produce at least a first ampliconcorresponding to the first microorganism and a second ampliconcorresponding to the second microorganism; (c) detecting the firstamplicon and the second amplicon produced in step (b) thereby generatinga first signal indicative of the first microorganism and a second signalindicative of the second microorganism; and (d) the method identifiesthe first microorganism by its genus or species using the first signaland the method identifies the second microorganism as eithergram-positive, gram-negative or fungal using the second signal.
 2. Themethod of claim 1, wherein i) the reagents for amplifying pan-targetsfor gram-negative bacteria, and ii) the reagents for amplifying speciesor genus targets for gram-positive bacteria, are in a first multiplexpool.
 3. The method of claim 2, wherein i) the reagents for amplifyingthe species targets for fungi and ii) the reagents for amplifyingspecies or genus targets for gram-positive bacteria are in a secondmultiplex pool.
 4. The method of claim 1, further comprising: loading asecond portion of the sample into a second cartridge, wherein the secondcartridge comprises reagents for amplifying species or genus targets forgram-negative bacteria or reagents for amplifying species or genustargets for fungi; amplifying nucleic acid in the second portion of thesample to produce a third amplicon corresponding to the secondmicroorganism; and detecting the third amplicon; thereby generating athird signal indicative of the second microorganism, wherein the methodidentifies the second microorganism by its genus or species using thethird signal.
 5. The method of claim 1, further comprising: loading asecond portion of the sample into a second cartridge, wherein the secondcartridge comprises reagents for amplifying species or genus targets forgram-positive bacteria or reagents for amplifying species or genustargets for fungi; amplifying nucleic acid in the second portion of thesample to produce a third amplicon corresponding to the secondmicroorganism; and detecting the third amplicon; thereby generating athird signal indicative of the second microorganism, wherein the methodidentifies the second microorganism by its genus or species using thethird signal.
 6. The method of claim 3, wherein the first and secondmultiplex pools are amplified in a single multiplex polymerase chainreaction (PCR) comprising about 30 to about 35 cycles.
 7. The method ofclaim 1, wherein the first signal and the second signal are detectedabove a threshold.
 8. The method of claim 1, wherein the first cartridgefurther comprises reagents for amplifying antibiotic resistance genes.9. The method of claim 1, wherein the first microorganism is aPropionibacterium acnes, a Staphylococcus epidermidis, a Micrococcus, aLactobacillus or a Corynebacterium.
 10. The method of claim 1, whereinprior to generating the first signal and the second signal, the firstamplicon and second amplicon are contacted with a plurality of signalprobes and a plurality of capture probes, wherein a first signal probeand a first capture probe are specific for the first amplicon, to form afirst hybridization complex, wherein a second signal probe and a secondcapture probe are specific for the second amplicon, to form a secondhybridization complex, and wherein the first hybridization complex andsecond hybridization complex are detected by electrochemical detection.11. The method of claim 1, wherein the identity of the firstmicroorganism and the second microorganism are reported to a hospitallaboratory information system.
 12. The method of claim 1, wherein atotal of about 60-90 minutes elapses from loading the sample into thefirst cartridge and identifying the species or genus of the firstmicroorganism.
 13. The method of claim 1, wherein the sample is obtainedfrom a subject with one or more symptoms of systemic inflammatoryresponse syndrome (SIRS), sepsis, severe sepsis or septic shock.
 14. Anin vitro method for identifying a first gram-positive bacterium thatinfects a subject by its species or genus and identifying a secondmicroorganism that infects a subject comprising: (a) loading a portionof a sample obtained from a gram-stain culture into a first cartridgewherein the gram stain culture identified the sample as comprising afirst gram-positive bacterium and wherein the first cartridge comprisesreagents for amplifying pan-targets for gram-negative bacteria, speciesor genus targets for gram-positive bacteria, and species targets forfungi; (b) amplifying nucleic acid in the portion of the sample toproduce at least a first amplicon corresponding to the firstgram-positive bacterium and a second amplicon corresponding to thesecond microorganism; (c) generating a first signal indicative of thefirst gram-positive bacterium and a second signal indicative of thesecond microorganism thereby detecting the first amplicon and the secondamplicon produced in step (b); and (d) the method identifies the firstgram-positive bacterium by its genus or species using the first signaland the method identifies the identity of the second microorganism aseither gram-negative or fungal using the second signal.
 15. The methodof claim 14, wherein the sample comprises viable and non-viablegram-positive bacteria.
 16. The method of claim 14, further comprising:loading a second portion of the sample into a second cartridge, whereinthe second cartridge comprises reagents for amplifying species or genustargets for gram-negative bacteria; amplifying nucleic acid in thesecond portion of the sample to produce a third amplicon correspondingto the second microorganism; and detecting the third amplicon andgenerating a third signal indicative of the second microorganism,wherein the method identifies the second microorganism by its genus orspecies using the third signal.
 17. The method of claim 14, furthercomprising: loading a second portion of the sample into a secondcartridge, wherein the second cartridge comprises reagents foramplifying species or genus targets for fungi; amplifying nucleic acidin the second portion of the sample to produce a third ampliconcorresponding to the second microorganism; and detecting the thirdamplicon and generating a third signal indicative of the secondmicroorganism, wherein the method identifies the second microorganism byits genus or species using the third signal.
 18. An in vitro method foridentifying a first gram-negative bacterium that infects a subject byits species or genus and identifying a second microorganism that infectsa subject comprising: (a) loading a portion of a sample obtained from agram-stain culture into a first cartridge wherein a gram stain cultureidentified the sample as comprising a first gram-negative bacterium andwherein the first cartridge comprises reagents for amplifying species orgenus targets for gram-positive bacteria, species or genus targets forgram-negative bacteria, and species targets for fungi; amplifyingnucleic acid in the portion of the sample to produce at least a firstamplicon corresponding to the first gram-negative bacterium and a secondamplicon corresponding to the second microorganism; (b) detecting thefirst amplicon and the second amplicon produced in step (Mb), therebygenerating a first signal indicative of the first gram-negativebacterium and a second signal indicative of the second microorganism;and (c) the method identifies the first gram-negative bacterium by itsgenus or species using the first signal and the method identifies thesecond microorganism as either gram-negative or fungal using the secondsignal.
 19. The method of claim 18, further comprising loading a secondportion of the sample into a second cartridge, wherein the secondcartridge comprises reagents for amplifying species or genus targets forgram-positive bacteria; amplifying nucleic acid in the sample to produceat least a third amplicon corresponding to the second microorganism; anddetecting the third amplicon; thereby generating a third signalindicative of the second microorganism; wherein the method identifiesthe second microorganism by its genus or species using the third signal.20. The method of claim 18, further comprising: loading a second portionof the sample into a second cartridge, wherein the second cartridgecomprises reagents for amplifying species or genus targets for fungi;amplifying nucleic acid in the second portion of the sample to produce athird amplicon corresponding to the second microorganism; and detectingthe third amplicon; thereby generating a third signal indicative of thesecond microorganism, wherein the method identifies the secondmicroorganism by its genus or species using the third signal.
 21. Themethod of claim 14, wherein the first amplicon and the second ampliconare detected using electrochemical detection to produce the first signaland the second signal, respectively.
 22. The method of claim 15, whereinthe viable gram-positive bacteria is present in the sample at a lowerconcentration than the non-viable gram-positive bacteria and the sampleis subject to a single detuned multiplex end-point polymerase chainreaction (PCR) to produce at least the first amplicon and the secondamplicon, the PCR comprising about 30 to about 35 cycles.
 23. The methodof claim 15, wherein the first amplicon corresponds to the viablegram-positive bacteria and is detected by electrochemical detection anda third amplicon, corresponding to the non-viable gram-positive bacteriais not detected by electrochemical detection.
 24. The method of claim14, further comprising, prior to step a, contacting the sample with acompound which hydrolyzes nucleic acids.
 25. The method of claim 14,wherein the viable gram-positive bacterium is a Bacillus cereus, aMicrococcus, a Bacillus subtilis, a Staphylococcus, a Staphylococcusaureus, a Propionibacterium acnes, a Staphylococcus epidermidis, aStaphylococcus lugdunensis, a Enterococcus faecalis, a Streptococcus, aEnterococcus faecium, a Streptococcus agalactiae, a Lactobacillus, aListeria, a Streptococcus pneumoniae, a Listeria monocytogenes, or aStreptococcus pyogenes.
 26. The method of claim 14, wherein step bfurther comprises amplifying nucleic acid in the portion of the sampleto produce at least a third amplicon corresponding to geneticdeterminants of resistance to methicillin or vancomycin; step c furthercomprises generating a third signal indicative of determinants ofresistance to methicillin or vancomycin, thereby detecting the thirdamplicon produced in step (b); and the method identifies the determinantof resistance to methicillin or vancomycin using the third signal. 27.The method of claim 14, wherein the method further comprisesautomatically generating and sending a detection report to a LISinterchange wherein the detection report comprises the identity of thefirst gram-positive bacterium by its genus or species and the identityof the second microorganism as either gram-negative or fungal.
 28. Themethod of claim 14, wherein the method further comprises automaticallygenerating and sending a quality control report to a LIS interchangewherein the quality control report comprises control data.
 29. Themethod of claim 14, wherein the method is carried out in asample-to-answer system wherein the sample-to-answer system isconfigured to (i) receive a test order from a hospital laboratoryinformation system (LIS), (ii) generate a detection report comprisingthe identity of the first gram-positive bacterium by its genus orspecies and identity of the second microorganism as either gram-negativeor fungal and (iii) automatically send the detection report to thehospital LIS.
 30. The method of claim 14, wherein the method is carriedout in a sample-to-answer system wherein the sample-to-answer system isconfigured to connect to more than one hospital laboratory informationsystem.